Methods of treating cancer using 3-(5-amino-2-methyl-4-oxo-4h-quinazolin-3-yl)-piperidine-2,6-dione

ABSTRACT

Provided herein are methods of treating, preventing and/or managing cancers, which comprise administering to a patient 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, or an enantiomer or a mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

This application claims the benefit of U.S. Provisional PatentApplication No. 61/451,995, filed on Mar. 11, 2011, and U.S. ProvisionalPatent Application No. 61/480,272, filed on Apr. 28, 2011, theentireties of which are incorporated herein by reference.

1. FIELD OF THE INVENTION

Provided herein are methods of treating, preventing and/or managingcancers, which comprise administering to a patient3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof.

2. BACKGROUND OF THE INVENTION

2.1 Pathobiology of Cancer

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, or lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia. Theneoplastic lesion may evolve clonally and develop an increasing capacityfor invasion, growth, metastasis, and heterogeneity, especially underconditions in which the neoplastic cells escape the host's immunesurveillance. Roitt, I., Brostoff, J and Kale, D., Immunology,17.1-17.12 (3rd ed., Mosby, St. Louis, Mo., 1993).

There is an enormous variety of cancers which are described in detail inthe medical literature. Examples include cancer of the lung, colon,rectum, prostate, breast, brain, and intestine. The incidence of cancercontinues to climb as the general population ages, as new cancersdevelop, and as susceptible populations (e.g., people infected with AIDSor excessively exposed to sunlight) grow. A tremendous demand thereforeexists for new methods and compositions that can be used to treatpatients with cancer.

Many types of cancers are associated with new blood vessel formation, aprocess known as angiogenesis. Several of the mechanisms involved intumor-induced angiogenesis have been elucidated. The most direct ofthese mechanisms is the secretion by the tumor cells of cytokines withangiogenic properties. Examples of these cytokines include acidic andbasic fibroblastic growth factor (a,b-FGF), angiogenin, vascularendothelial growth factor (VEGF), and TNF-α. Alternatively, tumor cellscan release angiogenic peptides through the production of proteases andthe subsequent breakdown of the extracellular matrix where somecytokines are stored (e.g., b-FGF). Angiogenesis can also be inducedindirectly through the recruitment of inflammatory cells (particularlymacrophages) and their subsequent release of angiogenic cytokines (e.g.,TNF-α, b-FGF).

Lymphoma refers to cancers that originate in the lymphatic system.Lymphoma is characterized by malignant neoplasms of lymphocytes—Blymphocytes and T lymphocytes (i.e., B-cells and T-cells). Lymphomagenerally starts in lymph nodes or collections of lymphatic tissue inorgans including, but not limited to, the stomach or intestines.Lymphoma may involve the marrow and the blood in some cases. Lymphomamay spread from one site to other parts of the body.

The treatment of various forms of lymphomas are described, for example,in U.S. Pat. No. 7,468,363, the entirety of which is incorporated hereinby reference. Such lymphomas include, but are not limited to, Hodgkin'slymphoma, non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, activatedB-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), follicular center lymphoma, transformed lymphoma,lymphocytic lymphoma of intermediate differentiation, intermediatelymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocyticlymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved celllymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Celllymphoma and mantle zone lymphoma and low grade follicular lymphoma.

Non-Hodgkin's lymphoma (NHL) is the fifth most common cancer for bothmen and women in the United States, with an estimated 63,190 new casesand 18,660 deaths in 2007. Jemal A, et al., CA Cancer J Clin 2007;57(1):43-66. The probability of developing NHL increases with age andthe incidence of NHL in the elderly has been steadily increasing in thepast decade, causing concern with the aging trend of the US population.Id. Clarke C A, et al., Cancer 2002; 94(7):2015-2023.

Diffuse large B-cell lymphoma (DLBCL) accounts for approximatelyone-third of non-Hodgkin's lymphomas. While some DLBCL patients arecured with traditional chemotherapy, the remainder die from the disease.Anticancer drugs cause rapid and persistent depletion of lymphocytes,possibly by direct apoptosis induction in mature T and B cells. See K.Stahnke. et al., Blood 2001, 98:3066-3073. Absolute lymphocyte count(ALC) has been shown to be a prognostic factor in follicularnon-Hodgkin's lymphoma and recent results have suggested that ALC atdiagnosis is an important prognostic factor in diffuse large B-celllymphoma. See D. Kim et al., Journal of Clinical Oncology, 2007 ASCOAnnual Meeting Proceedings Part I. Vol 25, No. 18S (June 20 Supplement),2007: 8082.

Leukemia refers to malignant neoplasms of the blood-forming tissues.Various forms of leukemias are described, for example, in U.S. Pat. No.7,393,862 and U.S. provisional patent application No. 60/380,842, filedMay 17, 2002, the entireties of which are incorporated herein byreference. Although viruses reportedly cause several forms of leukemiain animals, causes of leukemia in humans are to a large extent unknown.The Merck Manual, 944-952 (17^(th) ed. 1999). Transformation tomalignancy typically occurs in a single cell through two or more stepswith subsequent proliferation and clonal expansion. In some leukemias,specific chromosomal translocations have been identified with consistentleukemic cell morphology and special clinical features (e.g.,translocations of 9 and 22 in chronic myelocytic leukemia, and of 15 and17 in acute promyelocytic leukemia). Acute leukemias are predominantlyundifferentiated cell populations and chronic leukemias more mature cellforms.

Acute leukemias are divided into lymphoblastic (ALL) andnon-lymphoblastic (ANLL) types. The Merck Manual, 946-949 (17^(th) ed.1999). They may be further subdivided by their morphologic andcytochemical appearance according to the French-American-British (FAB)classification or according to their type and degree of differentiation.The use of specific B- and T-cell and myeloid-antigen monoclonalantibodies are most helpful for classification. ALL is predominantly achildhood disease which is established by laboratory findings and bonemarrow examination. ANLL, also known as acute myelogenous leukemia oracute myeloblastic leukemia (AML), occurs at all ages and is the morecommon acute leukemia among adults; it is the form usually associatedwith irradiation as a causative agent.

Chronic leukemias are described as being lymphocytic (CLL) or myelocytic(CML). The Merck Manual, 949-952 (17^(th) ed. 1999). CLL ischaracterized by the appearance of mature lymphocytes in blood, bonemarrow, and lymphoid organs. The hallmark of CLL is sustained, absolutelymphocytosis (>5,000/μL) and an increase of lymphocytes in the bonemarrow. Most CLL patients also have clonal expansion of lymphocytes withB-cell characteristics. CLL is a disease of middle or old age. In CML,the characteristic feature is the predominance of granulocytic cells ofall stages of differentiation in blood, bone marrow, liver, spleen, andother organs. In the symptomatic patient at diagnosis, the total whiteblood cell (WBC) count is usually about 200,000/μL, but may reach1,000,000/μL. CML is relatively easy to diagnose because of the presenceof the Philadelphia chromosome.

In addition to the acute and chronic categorization, neoplasms are alsocategorized based upon the cells giving rise to such disorder intoprecursor or peripheral. See e.g., U.S. patent publication no.2008/0051379, the disclosure of which is incorporated herein byreference in its entirety. Precursor neoplasms include ALLs andlymphoblastic lymphomas and occur in lymphocytes before they havedifferentiated into either a T- or B-cell. Peripheral neoplasms arethose that occur in lymphocytes that have differentiated into either T-or B-cells. Such peripheral neoplasms include, but are not limited to,B-cell CLL, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma,mantle cell lymphoma, follicular lymphoma, extranodal marginal zoneB-cell lymphoma of mucosa-associated lymphoid tissue, nodal marginalzone lymphoma, splenic marginal zone lymphoma, hairy cell leukemia,plasmacytoma, diffuse large B-cell lymphoma and Burkitt lymphoma. Inover 95 percent of CLL cases, the clonal expansion is of a B celllineage. See Cancer: Principles & Practice of Oncology (3rd Edition)(1989) (pp. 1843-1847). In less than 5 percent of CLL cases, the tumorcells have a T-cell phenotype. Notwithstanding these classifications,however, the pathological impairment of normal hematopoiesis is thehallmark of all leukemias.

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow.Normally, plasma cells produce antibodies and play a key role in immunefunction. However, uncontrolled growth of these cells leads to bone painand fractures, anemia, infections, and other complications. Multiplemyeloma is the second most common hematological malignancy, although theexact causes of multiple myeloma remain unknown. Multiple myeloma causeshigh levels of proteins in the blood, urine, and organs, including butnot limited to M-protein and other immunoglobulins (antibodies),albumin, and beta-2-microglobulin. M-protein, short for monoclonalprotein, also known as paraprotein, is a particularly abnormal proteinproduced by the myeloma plasma cells and can be found in the blood orurine of almost all patients with multiple myeloma.

Skeletal symptoms, including bone pain, are among the most clinicallysignificant symptoms of multiple myeloma. Malignant plasma cells releaseosteoclast stimulating factors (including IL-1, IL-6 and TNF) whichcause calcium to be leached from bones causing lytic lesions;hypercalcemia is another symptom. The osteoclast stimulating factors,also referred to as cytokines, may prevent apoptosis, or death ofmyeloma cells. Fifty percent of patients have radiologically detectablemyeloma-related skeletal lesions at diagnosis. Other common clinicalsymptoms for multiple myeloma include polyneuropathy, anemia,hyperviscosity, infections, and renal insufficiency.

Solid tumors are abnormal masses of tissue that may, but usually do notcontain cysts or liquid areas. Solid tumors may be benign (not cancer),or malignant (cancer). Different types of solid tumors are named for thetype of cells that form them. Examples of types solid tumors include,but are not limited to malignant melanoma, adrenal carcinoma, breastcarcinoma, renal cell cancer, carcinoma of the pancreas, non-small-celllung carcinoma (NSCLC) and carcinoma of unknown primary. Drugs commonlyadministered to patients with various types or stages of solid tumorsinclude, but are not limited to, celebrex, etoposide, cyclophosphamide,docetaxel, apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combinationthereof.

While patients who achieve a complete remission after initial therapyhave a good chance for cure, less than 10% of those who do not respondor relapse achieve a cure or a response lasting longer than 3 years. SeeCerny T, et al., Ann Oncol 2002; 13 Suppl 4:211-216.

Rituximab is known to deplete normal host B cells. See M. Aklilu et al.,Annals of Oncology 15:1109-1114, 2004. The long-term immunologic effectsof B cell depletion with rituximab and the characteristics of thereconstituting B cell pool in lymphoma patients are not well defined,despite the widespread usage of this therapy. See Jennifer H. Anolik etal., Clinical Immunology, vol. 122, issue 2, February 2007, pages139-145.

The approach for patients with relapsed or refractory disease reliesheavily on experimental treatments followed by stem celltransplantation, which may not be appropriate for patients with a poorperformance status or advanced age. Therefore, a tremendous demandexists for new methods that can be used to treat patients with NHL.

The link between cancer an altered cellular metabolism has been wellestablished. See Cairns, R. A., et al. Nature Rev., 2011, 11:85-95.Understanding tumor cell metabolism and the associated genetic changesthereof may lead to the identification of improved methods of cancertreatment. Id. For example, tumor cell survival and proliferation viaincreased glucose metabolism has been linked to the PIK3 pathway,whereby mutations in tumor suppressor genes such as PTEN activate tumorcell metabolism. Id. AKT1 (a.k.a., PKB) stimulates glucose metabolismassociated with tumor cell growth by various interactions with PFKFB3,ENTPD5, mTOR and TSC2 (a.k.a., tuberin). Id.

Transcription factors HIF1 and HIF2 are largely responsible for cellularresponse to low oxygen conditions often associated with tumors. Id. Onceactivated, HIF1 promotes tumor cell capacity to carry out glycolysis.Id. Thus, inhibition of HIF1 may slow or reverse tumor cell metabolism.Activation of HIF1 has been linked to PI3K, tumor suppressor proteinssuch as VHL, succinate dehydrogenase (SDH) and fumarate hydratase. Id.The oncogenic transcription factor MYC has also been linked to tumorcell metabolism, specifically glycolysis. Id. MYC also promotes cellproliferation by glutamine metabolic pathways. Id.

AMP-activated protein kinase (AMPK) functions as a metabolic check pointwhich tumor cells must overcome in order to proliferate. Id. Severalmutations have been identified which suppress AMPK signaling in tumorcells. See Shackelford, D. B. & Shaw, R. J., Nature Rev. Cancer, 2009,9: 563-575. STK11 has been identified as a tumor suppressor gene relatedto the role of AMPK. See Cairns, R. A., et al. Nature Rev., 2011,11:85-95.

The transcription factor p53, a tumor suppressor, also has an importantrole in the regulation of cellular metabolism. Id. The loss of p53 intumor cells may be a significant contributor to changes in tumor cellmetabolism to the glycolytic pathway. Id. The OCT1 transcription factor,another potential target for chemotherapeutics, may cooperate with p53in regulating tumor cell metabolism. Id.

Pyruvate kinate M2 (PKM2) promotes changes in cellular metabolism whichconfer metabolic advantages to cancer cells by supporting cellproliferation. Id. For example, lung cancer cells which express PKM2over PKM1 have been found to have such an advantage. Id. In the clinic,PKM2 has been identified as being overexpressed in a number of cancertypes. Id. Thus PKM2 may be a useful biomarker for the early detectionof tumors.

Mutations in isocitrate dehydrogenases IDH1 and IDH2 have been linked totumorigenesis, specifically, in glioblastoma and acute myeloid leukemia.See Mardis, E. R. et al., N. Engl. J. Med., 2009, 361: 1058-1066;Parsons, D. W. et al., Science, 2008, 321: 1807-1812.

The incidence of cancer continues to climb as the general populationages, as new cancers develop, and as susceptible populations (e.g.,people infected with AIDS, the elderly or excessively exposed tosunlight) grow. A tremendous demand therefore exists for new methods,treatments and compositions that can be used to treat patients withcancer including but not limited to those with lymphoma, NHL, multiplemyeloma, AML, leukemias, and solid tumors.

Accordingly, compounds that can control and/or inhibit unwantedangiogenesis or inhibit the production of certain cytokines, includingTNF-α, may be useful in the treatment and prevention of various forms ofcancer.

2.2 Methods of Treating Cancer

Current cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (see, for example, Stockdale, 1998, Medicine, vol. 3, Rubensteinand Federman, eds., Chapter 12, Section IV). Recently, cancer therapycould also involve biological therapy or immunotherapy. All of theseapproaches may pose significant drawbacks for the patient. Surgery, forexample, may be contraindicated due to the health of a patient or may beunacceptable to the patient. Additionally, surgery may not completelyremove neoplastic tissue. Radiation therapy is only effective when theneoplastic tissue exhibits a higher sensitivity to radiation than normaltissue. Radiation therapy can also often elicit serious side effects.Hormonal therapy is rarely given as a single agent. Although hormonaltherapy can be effective, it is often used to prevent or delayrecurrence of cancer after other treatments have removed the majority ofcancer cells. Certain biological and other therapies are limited innumber and may produce side effects such as rashes or swellings,flu-like symptoms, including fever, chills and fatigue, digestive tractproblems or allergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A number of cancerchemotherapeutics act by inhibiting DNA synthesis, either directly orindirectly by inhibiting the biosynthesis of deoxyribonucleotidetriphosphate precursors, to prevent DNA replication and concomitant celldivision. Gilman et al., Goodman and Gilman's: The Pharmacological Basisof Therapeutics, Tenth Ed. (McGraw Hill, New York).

Despite availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks. Stockdale, Medicine, vol. 3, Rubensteinand Federman, eds., ch. 12, sect. 10, 1998. Almost all chemotherapeuticagents are toxic, and chemotherapy causes significant and oftendangerous side effects including severe nausea, bone marrow depression,and immunosuppression. Additionally, even with administration ofcombinations of chemotherapeutic agents, many tumor cells are resistantor develop resistance to the chemotherapeutic agents. In fact, thosecells resistant to the particular chemotherapeutic agents used in thetreatment protocol often prove to be resistant to other drugs, even ifthose agents act by different mechanism from those of the drugs used inthe specific treatment. This phenomenon is referred to as multidrugresistance. Because of the drug resistance, many cancers proverefractory to standard chemotherapeutic treatment protocols.

There exists a significant need for safe and effective methods oftreating, preventing and managing cancer, particularly for cancers thatare refractory to standard treatments, such as surgery, radiationtherapy, chemotherapy and hormonal therapy, while reducing or avoidingthe toxicities and/or side effects associated with the conventionaltherapies.

2.3 Cereblon

The protein Cereblon (CRBN) is a 442-amino acid protein conserved fromplant to human. In humans, the CRBN gene has been identified as acandidate gene of an autosomal recessive nonsyndromic mental retardation(ARNSMR). See Higgins, J. J. et al., Neurology, 2004, 63:1927-1931. CRBNwas initially characterized as an RGS-containing novel protein thatinteracted with a calcium-activated potassium channel protein (SLO1) inthe rat brain, and was later shown to interact with a voltage-gatedchloride channel (CIC-2) in the retina with AMPK7 and DDB1. See Jo, S.et al., J. Neurochem, 2005, 94:1212-1224; Hohberger B. et al., FEBSLett, 2009, 583:633-637; Angers S. et al., Nature, 2006, 443:590-593.DDB1 was originally identified as a nucleotide excision repair proteinthat associates with damaged DNA binding protein 2 (DDB2). Its defectiveactivity causes the repair defect in the patients with xerodermapigmentosum complementation group E (XPE). DDB1 also appears to functionas a component of numerous distinct DCX (DDB1-CUL4-X-box) E3ubiquitin-protein ligase complexes which mediate the ubiquitination andsubsequent proteasomal degradation of target proteins. CRBN has alsobeen identified as a target for the development of therapeutic agentsfor diseases of the cerebral cortex. See WO 2010/137547 A1.

Cereblon has recently been identified as a key molecular target thatbinds to thalidomide to cause birth defects. See Ito, T. et al.,Science, 2010, 327:1345-1350. DDB1 was found to interact with CRBN and,thus, was indirectly associated with thalidomide. Moreover, thalidomidewas able to inhibit auto-ubiquitination of CRBN in vitro, suggestingthat thalidomide is an E3 ubiquitin-ligase inhibitor. Importantly, thisactivity was inhibited by thalidomide in wild-type cells, but not incells with mutated CRBN binding sites that prevent thalidomide binding.The thalidomide binding site was mapped to a highly conserved C-terminal104 amino acid region in CRBN. Individual point mutants in CRBN, Y384Aand W386A were both defective for thalidomide binding, with the doublepoint mutant having the lowest thalidomide-binding activity. A linkbetween CRBN and the teratogenic effect of thalidomide was confirmed inanimal models of zebra-fish and chick embryos. Understanding thalidomideand other drug targets will allow the definition of the molecularmechanisms of efficacy and/or toxicity and may lead to drugs withimproved efficacy and toxicity profiles.

3. SUMMARY OF THE INVENTION

Provided herein are methods of treating and preventing cancer, includingprimary and metastatic cancer, as well as cancer that is refractory orresistant to conventional chemotherapy, which comprise administering toa patient in need of such treatment or prevention a therapeutically orprophylactically effective amount of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,having the structure of Formula I:

or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof as a single agent or as a part of acombination therapy.

Also provided herein are methods of managing cancer (e.g., preventingits recurrence, or lengthening the time of remission), which compriseadministering to a patient in need of such management a therapeuticallyor prophylactically effective amount of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof.

Further provided herein are methods of treating, preventing, or managingcancer, comprising administering to a patient in need of such treatment,prevention, or management a therapeutically or prophylacticallyeffective amount of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof in combination with a therapy conventionally used to treat,prevent, or manage cancer. Examples of such conventional therapiesinclude, but are not limited to, surgery, chemotherapy, radiationtherapy, hormonal therapy, biological therapy, and immunotherapy.

Provided herein is a method for treating, preventing, or managingcancer, comprising administering to a patient in need of such treatment,prevention, or management3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, in an amount that is sufficient to provide a plasmaconcentration of the compound at steady state, of about 0.001 to about100 μM. In another embodiment, the amount is sufficient to provide apeak plasma concentration of the compound at steady state, of about0.001 to about 100 μM. In another embodiment, the amount is sufficientto provide a trough plasma concentration of the compound at steadystate, of about 0.01 to about 100 μM. In another embodiment, the amountis sufficient to provide an area under the curve (AUC) of the compound,ranging from about 100 to about 100,000 ng*hr/mL.

In certain embodiments, provided herein are methods for the treatment ormanagement of lymphoma, multiple myeloma, leukemia, and solid tumors.

In some embodiments, the lymphoma is selected from the group consistingof Hodgkin's lymphoma, non-Hodgkin's lymphoma, AIDS-related lymphomas,anaplastic large-cell lymphoma, angioimmunoblastic lymphoma, blasticNK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (smallnon-cleaved cell lymphoma, small lymphocytic lymphoma, cutaneous T-celllymphoma, diffuse large B-cell Lymphoma, enteropathy-type T-celllymphoma, lymphoblastic lymphoma, mantle cell lymphoma, marginal zonelymphoma, nasal T-cell lymphoma, pediatric lymphoma, peripheral T-celllymphomas, primary central nervous system lymphoma, transformedlymphomas, treatment-related T-cell lymphomas and Waldenstrom'smacroglobulinemia.

In some embodiments, the leukemia is selected from the group consistingof acute myeloid leukemia (AML), T-cell leukemia, chronic myeloidleukemia (CML), chronic lymphocytic leukemia (CLL) and acutllymphoblastic leukemia (ALL).

In some embodiments, the solid tumor is selected from the groupconsisting of melanoma, head and neck tumors, breast carcinoma,non-small cell lung carcinoma, ovarian carcinoma, pancreatic carcinoma,prostate carcinoma, colorectal carcinoma, and hepatocellular carcinoma.

In some embodiments, provided herein are methods for the treatment ormanagement of non-Hodgkin's lymphomas, including but not limited to,diffuse large B-cell lymphoma (DLBCL), using prognostic factors.

In some embodiments, provided herein are methods for the use of gene andprotein biomarkers as a predictor of clinical sensitivity to lymphoma,non-Hodgkin's lymphoma, multiple myeloma, leukemia, AML, and/or solidtumors and patient response to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof.

The methods provided herein encompass methods for screening oridentifying cancer patients, e.g., lymphoma, non-Hodgkin's lymphoma,multiple myeloma, leukemia, AML, and solid tumor patients, for treatmentwith 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof. In particular, provided herein aremethods for selecting patients having a higher response rate to therapywith 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione.

In one embodiment, provided herein is a method of predicting tumorresponse to treatment in a lymphoma, non-Hodgkin's lymphoma, multiplemyeloma, leukemia, AML or solid tumor patient, the method comprisingobtaining tumor tissue from the patient, purifying protein or RNA fromthe tumor, and measuring the presence or absence of a biomarker by,e.g., protein or gene expression analysis. The expression monitored maybe, for example, mRNA expression or protein expression.

In certain embodiments, the biomarker is a gene associated with anactivated B-cell phenotype of DLBCL. The genes are selected from thegroup consisting of IRF4/MUM1, FOXP1, SPIB, CARD11 and BLIMP/PDRM1. Inone embodiment, the biomarker is NF-κB.

In one embodiment, the mRNA or protein is purified from the tumor andthe presence or absence of a biomarker is measured by gene or proteinexpression analysis. In certain embodiments, the presence or absence ofa biomarker is measured by quantitative real-time PCR (QRT-PCR),microarray, flow cytometry or immunofluorescence. In other embodiments,the presence or absence of a biomarker is measured by enzyme-linkedimmunosorbent assay-based methodologies (ELISA) or other similar methodsknown in the art.

In another embodiment, provided herein is a method of predicting tumorresponse to treatment in a non-Hodgkin's lymphoma patient, the methodcomprising obtaining tumor cells from the patient, culturing the cellsin the presence or absence of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,purifying protein or RNA from the cultured cells, and measuring thepresence or absence of a biomarker by, e.g., protein or gene expressionanalysis. The expression monitored may be, for example, mRNA expressionor protein expression.

In another embodiment, provided herein is a method of monitoring tumorresponse to3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionetreatment in a lymphoma, non-Hodgkin's lymphoma, multiple myeloma,leukemia, AML, or solid tumor patient. The method comprises obtaining abiological sample from the patient, measuring the expression of abiomarker in the biological sample, administering3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione tothe patient, thereafter obtaining a second biological sample from thepatient, measuring biomarker expression in the second biological sample,and comparing the levels of expression, where an increased level ofbiomarker expression after treatment indicates the likelihood of aneffective tumor response. In one embodiment, a decreased level ofbiomarker expression after treatment indicates the likelihood ofeffective tumor response. The biomarker expression monitored can be, forexample, mRNA expression or protein expression. The expression in thetreated sample can increase, for example, by about 1.5×, 2.0×, 3×, 5×,or more.

In yet another embodiment, a method for monitoring patient compliancewith a drug treatment protocol is provided. The method comprisesobtaining a biological sample from the patient, measuring the expressionlevel of at least one biomarker in the sample, and determining if theexpression level is increased or decreased in the patient samplecompared to the expression level in a control untreated sample, whereinan increased or decreased expression indicates patient compliance withthe drug treatment protocol. In one embodiment, the expression of one ormore biomarkers is increased. The biomarker expression monitored can be,for example, mRNA expression or protein expression. The expression inthe treated sample can increase, for example, by about 1.5×, 2.0×, 3×,5×, or more.

In another embodiment, provided herein is a method of predicting thesensitivity to treatment3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione in alymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia, AML orsolid tumor patient. In one embodiment, the patient is a non-Hodgkin'slymphoma patient, specifically, a DLBCL patient. The method comprisesobtaining a biological sample from the patient, optionally isolating orpurifying mRNA from the biological sample, amplifying the mRNAtranscripts by, e.g., RT-PCR, where a higher baseline level of aspecific biomarker indicates a higher likelihood that the cancer will besensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Incertain embodiments, the biomarker is a gene associated with anactivated B-cell phenotype. The genes are selected from the groupconsisting of IRF4/MUM1, FOXP1, SPIB, CARD11 and BLIMP/PDRM1.

Also provided herein are methods for the treatment or management ofcancer with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione usingCRBN as a predictive or prognostic factor. In certain embodiments,provided herein are methods for screening or identifying cancer patientsfor treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione usingCRBN levels as a predictive or prognostic factor. In some embodiments,provided herein are methods for selecting patients having a higherresponse rate to therapy with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione usingCRBN levels as a predictive or prognostic factor.

In one embodiment, provided herein is a method of predicting patientresponse to treatment of cancer with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, themethod comprising obtaining biological material from the patient, andmeasuring the presence or absence of CRBN.

In one embodiment, the method comprises obtaining cancer cells from thepatient, culturing the cells in the presence or absence of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,purifying protein or RNA from the cultured cells, and measuring thepresence or absence of a biomarker by, e.g., protein or gene expressionanalysis. The expression monitored may be, for example, mRNA expressionor protein expression. In one embodiment, the cancer is lymphoma,leukemia, multiple myeloma, solid tumor, non-Hodgkin's lymphoma ormelanoma.

In another embodiment, provided herein is a method of monitoring tumorresponse to drug treatment in a cancer patient. The method comprisesobtaining a biological sample from the patient, measuring the expressionof a biomarker in the biological sample, administering one or more drugsto the patient, thereafter obtaining a second biological sample from thepatient, measuring biomarker expression in the second biological sample,and comparing the levels of expression, where an increased level ofbiomarker expression after treatment indicates the likelihood of aneffective tumor response. In one embodiment, the cancer patient is alymphoma, leukemia, multiple myeloma, solid tumor, non-Hodgkin'slymphoma or melanoma patient.

In one embodiment, a decreased level of biomarker expression aftertreatment indicates the likelihood of effective tumor response. Thebiomarker expression monitored can be, for example, mRNA expression orprotein expression. The expression in the treated sample can increase,for example, by about 1.5×, 2.0×, 3×, 5×, or more. In one embodiment,the tumor is a lymphoma, leukemia, multiple myeloma, solid tumor,non-Hodgkin's lymphoma or melanoma.

In another embodiment, provided herein is a method of predicting thesensitivity to drug treatment in a cancer patient, specifically, amultiple myeloma or non-Hodgkin's lymphoma patient. The method comprisesobtaining a biological sample from the patient, optionally isolating orpurifying mRNA from the biological sample, amplifying the mRNAtranscripts by, e.g., RT-PCR, where a higher baseline level of aspecific biomarker indicates a higher likelihood that the cancer will besensitive to treatment with a drug. In certain embodiments, thebiomarker is a gene or protein associated with multiple myeloma ornon-Hodgkin's lymphoma. In one embodiment, the genes are thoseassociated with CRBN and are selected from the group consisting of DDB1,DDB2, GSK3B, CUL4A, CUL4B, XBP-1, FAS1, RANBP6, DUS3L, PHGDH, AMPK, IRF4and NFκB. In another embodiment, the genes are selected from the groupconsisting of DDB1, DDB2, IRF4 and NFκB.

In one embodiment, identifying a patient having lymphoma, leukemia,multiple myeloma, a solid tumor, non-Hodgkin's lymphoma, diffuse largeB-cell lymphoma or melanoma sensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione;identification of a gene or protein associated with CRBN wherein thepresence of the gene or protein associated with CRBN is indicative oflymphoma, leukemia, multiple myeloma, a solid tumor, non-Hodgkin'slymphoma, diffuse large B-cell lymphoma or melanoma sensitive totreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Inone embodiment, the gene or protein associated with CRBN is selectedfrom the group consisting of DDB1, DDB2, IRF4 and NFκB.

In one embodiment, identifying a patient having lymphoma, leukemia,multiple myeloma, a solid tumor, non-Hodgkin's lymphoma or melanomasensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises measuring the level of CRBN activity in the patient. Inanother embodiment, measuring the level of CRBN activity in the patientcomprises measuring DDB1, DDB2, IRF4 and/or NFκB in cells obtained fromthe patient.

In one embodiment, provided herein is a method for treating or managingnon-Hodgkin's lymphoma, comprising:

(i) identifying a patient having lymphoma, non-Hodgkin's lymphoma,multiple myeloma, leukemia, AML or a solid tumor sensitive to treatmentwith 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione;and

(ii) administering to the patient a therapeutically effective amount of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, or apharmaceutically acceptable salt or solvate (e.g., hydrate) thereof.

In one embodiment, the patient has non-Hodgkin's lymphoma. In oneembodiment, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.In another embodiment, the non-Hodgkin's lymphoma is of the activatedB-cell phenotype.

In one embodiment, identifying a patient having non-Hodgkin's lymphomasensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises identification of a gene associated with the activated B-cellphenotype. In one embodiment, the gene associated with the activatedB-cell phenotype is selected from the group consisting of IRF4/MUM1,FOXP1, SPIB, CARD11 and BLIMP/PDRM1.

In one embodiment, identifying a patient having non-Hodgkin's lymphomasensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises measuring the level of NF-κB activity in the patient. Inanother embodiment, measuring the level of NF-κB activity in the patientcomprises measuring the baseline NF-κB activity level in tumor cellsobtained from the patient.

Also provided herein are kits useful for predicting the likelihood of aneffective lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia,AML or solid tumor treatment or for monitoring the effectiveness of atreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Thekit comprises a solid support, and a means for detecting the proteinexpression of at least one biomarker in a biological sample. Such a kitmay employ, for example, a dipstick, a membrane, a chip, a disk, a teststrip, a filter, a microsphere, a slide, a multiwell plate, or anoptical fiber. The solid support of the kit can be, for example, aplastic, silicon, a metal, a resin, glass, a membrane, a particle, aprecipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, acapillary, a film, a plate, or a slide. The biological sample can be,for example, a cell culture, a cell line, a tissue, an oral tissue,gastrointestinal tissue, an organ, an organelle, a biological fluid, ablood sample, a urine sample, or a skin sample. The biological samplecan be, for example, a lymph node biopsy, a bone marrow biopsy, or asample of peripheral blood tumor cells.

In an additional embodiment, provided herein is a kit useful forpredicting the likelihood of an effective treatment or for monitoringthe effectiveness of a treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Thekit comprises a solid support, nucleic acids contacting the support,where the nucleic acids are complementary to at least 20, 50, 100, 200,350, or more bases of mRNA, and a means for detecting the expression ofthe mRNA in a biological sample.

In another embodiment, provided herein is a kit useful for predictingthe likelihood of an effective treatment or for monitoring theeffectiveness of a treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Thekit comprises a solid support, at least one nucleic acid contacting thesupport, where the nucleic acid is complementary to at least 20, 50,100, 200, 350, 500, or more bases of mRNA, and a means for detecting theexpression of the mRNA in a biological sample.

In certain embodiments, the kits provided herein employ means fordetecting the expression of a biomarker by quantitative real-time PCR(QRT-PCR), microarray, flow cytometry or immunofluorescence. In otherembodiments, the expression of the biomarker is measured by ELISA-basedmethodologies or other similar methods known in the art.

Also provided herein are pharmaceutical compositions comprising about 1to 1,000 mg of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof.

Further provided herein are pharmaceutical compositions comprising about1 to 1,000 mg of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof; and one or more additional active ingredient. In certainembodiments, the one or more additional active ingredients are selectedfrom oblimersen, melphalan, G-CSF, GM-CSF, EPO, a cox-2 inhibitor,topotecan, pentoxifylline, ciprofloxacin, taxotere, iritotecan,dexamethasone, doxorubicin, vincristine, IL 2, IFN, dacarbazine, Ara-C,vinorelbine and isotretinoin.

Also provided herein are kits useful for predicting the likelihood of aneffective lymphoma, leukemia, multiple myeloma, a solid tumor,non-Hodgkin's lymphoma, diffuse large B-cell lymphoma or melanomatreatment or for monitoring the effectiveness of a treatment with one ormore drugs, e.g.,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Thekit comprises a solid support, and a means for detecting the proteinexpression of at least one biomarker in a biological sample. Such a kitmay employ, for example, a dipstick, a membrane, a chip, a disk, a teststrip, a filter, a microsphere, a slide, a multiwell plate, or anoptical fiber. The solid support of the kit can be, for example, aplastic, silicon, a metal, a resin, glass, a membrane, a particle, aprecipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, acapillary, a film, a plate, or a slide. The biological sample can be,for example, a cell culture, a cell line, a tissue, an oral tissue,gastrointestinal tissue, an organ, an organelle, a biological fluid, ablood sample, a urine sample, or a skin sample. The biological samplecan be, for example, a lymph node biopsy, a bone marrow biopsy, or asample of peripheral blood tumor cells.

In another embodiment, the kit comprises a solid support, nucleic acidscontacting the support, where the nucleic acids are complementary to atleast 20, 50, 100, 200, 350, or more bases of mRNA, and a means fordetecting the expression of the mRNA in a biological sample.

In certain embodiments, the kits provided herein employ means fordetecting the expression of a biomarker by quantitative real-time PCR(QRT-PCR), microarray, flow cytometry or immunofluorescence. In otherembodiments, the expression of the biomarker is measured by ELISA-basedmethodologies or other similar methods known in the art.

Also provided herein is a kit comprising (i) a pharmaceuticalcomposition comprising3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof; and (ii) a pharmaceutical composition comprising hematopoieticgrowth factor, cytokine, anti-cancer agent, antibiotic, a cox-2inhibitor, immunomodulatory agent, immunosuppressive agent,corticosteroid, or a pharmacologically active mutant or derivativethereof, or a combination thereof.

In one embodiment, provided herein is a kit comprising (i) apharmaceutical composition comprising3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof; and (ii) a pharmaceutical composition comprising oblimersen,melphalan, G-CSF, GM-CSF, EPO, a cox-2 inhibitor, topotecan,pentoxifylline, taxotere, iritotecan, ciprofloxacin, dexamethasone,doxorubicin, vincristine, IL 2, IFN, dacarbazine, Ara-C, vinorelbine, orisotretinoin.

In another embodiment, provided herein is a kit comprising (i) apharmaceutical composition comprising3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof; and (ii) umbilical cord blood, placental blood, peripheralblood stem cell, hematopoietic stem cell preparation or bone marrow.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1D: Inhibitory effect of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione(Comp. Formula I) on NF-κB activity in DLBCL cells.

FIG. 2: Antiproliferative effect of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione(Compound of Formula I) in an in vitro DLBCL cell-based assay.

FIG. 3:3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione(Compound of Formula I) costimulates T cells and enhances cytokineproduction.

FIG. 4: Anti-angiogenic effect of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionein anin vitro human umbilical expant assay.

FIGS. 5A & 5B: Antiproliferative effect of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione in anin vitro multiple myleoma (MM) cell-based assay.

FIG. 6: In vitro tumor inhibition of Antiproliferative effect of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione in aN929 xenograft model.

FIGS. 7A-7C: Cereblon expression modulates the effects of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione inABC-DLBCL cell lines.

FIG. 8: Knockdown of CRBN abrogated G1 arrest induced by3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione.

FIGS. 9A-9F: CRBN knockdown abrogates effect of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione onphosphorylation of pRb and IRF-4 in H929 cells.

FIG. 10: Antiproliferative activity of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dioneinhibits in CRBN-sensitive myeloma cells.

FIG. 11: Cereblon expression modulates anti-invasive activity of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione.

FIGS. 12A-12I:3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dioneinhibits hypoxia-induced HIF1-α expression in solid tumor cell lines.

FIGS. 13A & 13B:3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dioneinhibits breast cancer cell colony formation.

FIG. 14:3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dioneinhibits U87 glioiblastoma tumor cell growth.

5. DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods of treating, managing, or preventing cancer,which comprise administering to a patient in need of such treatment,management, or prevention a therapeutically or prophylacticallyeffective amount of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof as a single agent or as a part of a combination therapy.

In certain embodiments,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, is administered in combination with one or more additionaldrugs (or “second active agents”) for use in the treatment, management,or prevention of cancer. Second active agents include small moleculesand large molecules (e.g., proteins and antibodies), some examples ofwhich are provided herein, as well as stem cells. Methods or therapies,that can be used in combination with the administration of the compoundprovided herein include, but are not limited to, surgery, bloodtransfusions, immunotherapy, biological therapy, radiation therapy, andother non-drug based therapies presently used to treat, prevent ormanage cancer. In certain embodiments, the compound provided herein maybe used as a vaccine adjuvant.

In some embodiments, the methods provided herein are based, in part, onthe discovery that the expression of certain genes or proteinsassociated with certain cancer cells may be utilized as biomarkers toindicate the effectiveness or progress of a disease treatment. Suchcancers include, but are not limited to, lymphoma, non-Hodgkin'slymphoma, multiple myeloma, leukemia, acute myeloid leukemia (AML), andsolid tumors. In certain embodiments, the cancer is of the activatedB-cell phenotype in non-Hodgkin's lymphoma. In particular, thesebiomarkers can be used to predict, assess and track the effectiveness ofpatient treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione.

In some embodiments, the methods provided herein are based, in part, onthe discovery that cereblon (CRBN) is associated with theanti-proliferative activities of certain drugs, such as3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Insome embodiments, CRBN may be utilized as a biomarker to indicate theeffectiveness or progress of a disease treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione.Without being bound by a particular theory, CRBN binding may contributeto or even be required for anti-proliferative or other activities ofcertain compounds, such as3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione

Without being limited to a particular theory,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione canmediate growth inhibition, apoptosis and inhibition of angiogenicfactors in certain types of cancer such as lymphoma, non-Hodgkin'slymphoma, multiple myeloma, leukemia, AML, and solid tumors. Uponexamining the expression of several cancer-related genes in several celltypes before and after the treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, itwas discovered that the expression levels of several cancer-relatedgenes or proteins can be used as biomarkers for predicting andmonitoring cancer treatments.

It was also discovered that the level of NF-κB activity is elevated incells of the activated B-cell phenotype in non-Hodgkin's lymphomarelative to other types of lymphoma cells, and that such cells may besensitive to3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionetreatment. This suggests that the baseline activity of NF-κB activity inlymphoma cells may be a predictive biomarker for3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionetreatment in non-Hodgkin's lymphoma patients.

Therefore, in certain embodiments, provided herein are methods forpredicting tumor response to treatment in a non-Hodgkin's lymphomapatient. In one embodiment, provided herein is a method of predictingtumor response to treatment in a non-Hodgkin's lymphoma patient, themethod comprising obtaining tumor tissue from the patient, purifyingprotein or RNA from the tumor, and measuring the presence or absence ofa biomarker by, e.g., protein or gene expression analysis. Theexpression monitored may be, for example, mRNA expression or proteinexpression. In certain embodiments, the biomarker is a gene associatedwith an activated B-cell phenotype of DLBCL. The genes are selected fromthe group consisting of IRF4/MUM1, FOXP1, SPIB, CARD11 and BLIMP/PDRM1.In one embodiment, the biomarker is NF-κB.

In another embodiment, the method comprises obtaining tumor cells fromthe patient, culturing the cells in the presence or absence of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,purifying RNA or protein from the cultured cells, and measuring thepresence or absence of a biomarker by, e.g., gene or protein expressionanalysis.

In certain embodiments, the presence or absence of a biomarker ismeasured by quantitative real-time PCR (QRT-PCR), microarray, flowcytometry or immunofluorescence. In other embodiments, the presence orabsence of a biomarker is measured by ELISA-based methodologies or othersimilar methods known in the art.

The methods provided herein encompass methods for screening oridentifying cancer patients, e.g., non-Hodgkin's lymphoma patients, fortreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Inparticular, provided herein are methods for selecting patients having,or who are likely to have, a higher response rate to a therapy with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione.

In one embodiment, the method comprises the identification of patientslikely to respond to therapy by obtaining tumor cells from the patient,culturing the cells in the presence or absence of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,purifying RNA or protein from the cultured cells, and measuring thepresence or absence of a specific biomarker. The expression monitoredcan be, for example, mRNA expression or protein expression. Theexpression in the treated sample can increase, or in some cases,decrease, for example, by about 1.5×, 2.0×, 3×, 5×, or more. In certainembodiments, the biomarker is a gene associated with an activated B-cellphenotype. The genes are selected from the group consisting ofIRF4/MUM1, FOXP1, SPIB, CARD11 and BLIMP/PDRM1. In one embodiment, thebiomarker is NF-κB. Baseline levels of expression of these genes can bepredictive of the sensitivity of a cancer to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione.

In one embodiment, IRF4/MUM1 expression in cancer cells, e.g.,ABC-subtype lymphoma, can be decreased with the treatment of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Insome embodiments, IRF4 downregulation by3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione canbe a potential pharmacodynamic biomarker.

In another embodiment, provided herein is a method of monitoring tumorresponse to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione in alymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia, AML or asolid tumor patient. The method comprises obtaining a biological samplefrom the patient, measuring the expression of one or more biomarkers inthe biological sample, administering3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione tothe patient, thereafter obtaining a second biological sample from thepatient, measuring biomarker expression in the second biological sample,and comparing the levels of biomarker expression, where an increasedlevel of biomarker expression after treatment indicates the likelihoodof an effective tumor response. In one embodiment, a decreased level ofbiomarker expression after treatment indicates the likelihood ofeffective tumor response. In certain embodiments, the biomarker is agene associated with an activated B-cell phenotype of non-Hodgkin'slymphoma. The genes are selected from the group consisting of IRF4/MUM1,FOXP1, SPIB, CARD11 and BLIMP/PDRM1. In one embodiment, the biomarker isNF-κB.

In certain embodiments, the method comprises measuring the expression ofone or more biomarkers genes associated with an activated B-cellphenotype. The genes are selected from the group consisting ofIRF4/MUM1, FOXP1, SPIB, CARD11 and BLIMP/PDRM1. The expression monitoredcan be, for example, mRNA expression or protein expression. Theexpression in the treated sample can increase, for example, by about1.5×, 2.0×, 3×, 5×, or more.

In yet another embodiment, a method for monitoring patient compliancewith a drug treatment protocol is provided. The method comprisesobtaining a biological sample from the patient, measuring the expressionlevel of at least one biomarker in the sample, and determining if theexpression level is increased or decreased in the patient samplecompared to the expression level in a control untreated sample, whereinan increased or decreased expression indicates patient compliance withthe drug treatment protocol. In one embodiment, the expression of one ormore biomarker is increased. The expression monitored can be, forexample, mRNA expression or protein expression. The expression in thetreated sample can increase, for example, by about 1.5×, 2.0×, 3×, 5×,or more. In certain embodiments, the biomarker is a gene associated withan activated B-cell phenotype. The genes are selected from the groupconsisting of IRF4/MUM1, FOXP1, SPIB, CARD11 and BLIMP/PDRM1. In oneembodiment, the biomarker is NF-κB.

In another embodiment, a method of predicting the sensitivity totreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione in alymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia, AML or asolid tumor patient is provided. In one embodiment, the patient is anon-Hodgkin's lymphoma patient, specifically, a DLBCL patient. Themethod comprises obtaining a biological sample from the patient,optionally isolating or purifying mRNA from the biological sample,amplifying the mRNA transcripts by, e.g., RT-PCR, where a higherbaseline level of one or more specific biomarkers indicates a higherlikelihood that the cancer will be sensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Inone embodiment, the biomarker is a gene associated with an activatedB-cell phenotype selected from the group consisting of IRF4/MUM1, FOXP1,SPIB, CARD11 and BLIMP/PDRM1.

In another embodiment, the method of predicting sensitivity to treatmentwith 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionein an NHL, e.g., a DLBCL patient, comprises obtaining a tumor samplefrom the patient, embedding the tumor sample into a paraffin-embedded,formalin-fixed block, and staining the sample with antibodies to CD20,CD10, bcl-6, IRF4/MUM1, bcl-2, cyclin D2, and/or FOXP1, as described inHans et al., Blood, 2004, 103: 275-282, which is hereby incorporated byreference in its entirety. In one embodiment, CD10, bcl-6, andIRF4/MUM-1 staining can be used to divide DLBCL into GCB and non-GCBsubgroups to predict an outcome.

In one embodiment, provided herein is a method for predicting tumorresponse to treatment in a non-Hodgkin's lymphoma patient, comprising:

(i) obtaining a biological sample from the patient;(ii) measuring activity of the NF-κB pathway in the biological sample;and(iii) comparing the level of NF-κB activity in the biological sample tothat of a biological sample of a non-activated B-cell lymphoma subtype;

wherein an increased level of NF-κB activity relative to non-activatedB-cell subtype lymphoma cells indicates a likelihood of an effectivepatient tumor response to3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionetreatment.

In one embodiment, measuring activity of the NF-κB pathway in thebiological sample comprises measuring the level of NF-κB in thebiological sample.

In one embodiment, provided herein is a method of monitoring tumorresponse to treatment in a non-Hodgkin's lymphoma patient, comprising:

(i) obtaining a biological sample from the patient;(ii) measuring the level of NF-κB activity in the biological sample;(iii) administering a therapeutically effective amount of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, or asalt, solvate or hydrate thereof to the patient;(iv) obtaining a second biological sample from the patient;(v) measuring the level of NF-κB activity in the second biologicalsample; and(vi) comparing the level of NF-κB activity in the first biologicalsample to that in the second biological sample;

wherein a decreased level of NF-κB activity in the second biologicalsample relative to the first biological sample indicates a likelihood ofan effective patient tumor response.

In one embodiment, provided herein is a method for monitoring patientcompliance with a drug treatment protocol in a non-Hodgkin's lymphomapatient, comprising:

(i) obtaining a biological sample from the patient;(ii) measuring the level of NF-κB activity in the biological sample; and(iii) comparing the level of NF-κB activity in the biological sample toa control untreated sample;

wherein a decreased level of NF-κB activity in the biological samplerelative to the control indicates patient compliance with the drugtreatment protocol.

In one embodiment, the non-Hodgkin's lymphoma is diffuse large B-celllymphoma.

In another embodiment, the level of NF-κB activity is measured by anenzyme-linked immunosorbent assay.

In one embodiment, provided herein is a method for predicting tumorresponse to treatment in a non-Hodgkin's lymphoma patient, comprising:

(i) obtaining a biological sample from the patient;(ii) culturing cells from the biological sample;(iii) purifying RNA from the cultured cells; and(iv) identifying increased expression of a gene associated with theactivated B-cell phenotype of non-Hodgkin's lymphoma relative to controlnon-activated B-cell phenotype of non-Hodgkin's lymphoma;

wherein increased expression of a gene associated with the activatedB-cell phenotype of non-Hodgkin's lymphoma indicates a likelihood of aneffective patient tumor response to3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionetreatment.

In one embodiment, increased expression is an increase of about 1.5×,2.0×, 3×, 5×, or more.

In one embodiment, the gene associated with the activated B-cellphenotype is selected from the group consisting of IRF4/MUM1, FOXP1,SPIB, CARD11 and BLIMP/PDRM1.

In one embodiment, identifying the expression of a gene associated withthe activated B-cell phenotype of non-Hodgkin's lymphoma is performed byquantitative real-time PCR.

Also provided herein is a method for treating or managing non-Hodgkin'slymphoma, comprising:

(i) identifying a patient having non-Hodgkin's lymphoma sensitive totreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione; and

(ii) administering to the patient a therapeutically effective amount of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, or apharmaceutically acceptable salt, solvate or hydrate thereof.

In one embodiment, the non-Hodgkin's lymphoma is diffuse large B-celllymphoma.

In another embodiment, the non-Hodgkin's lymphoma is of the activatedB-cell phenotype.

In another embodiment, the diffuse large B-cell lymphoma ischaracterized by the expression of one or more biomarkers overexpressedin RIVA, U2932, TMD8, OCI-Ly3 or OCI-Ly10 cell lines.

In another embodiment, the diffuse large B-cell lymphoma ischaracterized by the expression of one or more biomarkers overexpressedin RIVA, U2932, TMD8 or OCI-Ly10 cell lines.

In one embodiment, identifying a patient having lymphoma sensitive totreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises characterization of the lymphoma phenotype of the patient.

In one embodiment, the lymphoma phenotype is characterized as anactivated B-cell subtype.

In one embodiment, the lymphoma phenotype is characterized as anactivated B-cell subtype of diffuse large B-cell lymphoma.

In certain embodiments, identification of the lymphoma phenotypecomprises obtaining a biological sample from a patient having lymphoma.In one embodiment, the biological sample is a cell culture or tissuesample. In one embodiment, the biological sample is a sample of tumorcells. In another embodiment, the biological sample is a lymph nodebiopsy, a bone marrow biopsy, or a sample of peripheral blood tumorcells. In one embodiment, the biological sample is a blood sample.

In one embodiment, identifying a patient having non-Hodgkin's lymphomasensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises identification of a gene associated with an activated B-cellphenotype. In one embodiment, the gene associated with the activatedB-cell phenotype is selected from the group consisting of IRF4/MUM1,FOXP1, SPIB, CARD11 and BLIMP/PDRM1.

In one embodiment, identifying a patient having non-Hodgkin's lymphomasensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dionecomprises measuring the level of NF-κB activity in the patient. Inanother embodiment, measuring the level of NF-κB activity in a patientcomprises measuring the baseline NF-κB activity level in tumor cellsobtained from the patient.

In another embodiment, the diffuse large B-cell lymphoma ischaracterized by one or more of the following:

(i) over expression of SPIB, a hematopoietic-specific Ets familytranscription factor required for survival of activated B-cell subtypecells;(ii) higher constitutive IRF4/MUM1 expression than GCB subtype cells;(iii) higher constitutive FOXP1 expression up-regulated by trisomy 3;(iv) higher constitutive Blimp 1, i.e., PRDM1, expression; and(v) higher constitutive CARD11 gene expression; and(vi) an increased level of NF-κB activity relative to non-activatedB-cell subtype DLBCL cells.

Additional prognostic factors that may be used concurrently with thoseprovided herein are prognostic factors of disease (tumor) burden,absolute lymphocyte count (ALC), time since last rituximab therapy forlymphomas, or all of the above.

Also provided herein is a method of selecting a group of cancer patientsbased on the level of CRBN expression, or the levels of DDB1, DDB2, IRF4or NF-κB expression within the cancer, for the purposes of predictingclinical response, monitoring clinical response, or monitoring patientcompliance to dosing by3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof; wherein the cancerpatients are selected from multiple myeloma, non-Hodgkin's lymphoma,diffuse large B-cell lymphoma, melanoma and solid tumor patients.Baseline levels of expression of these genes can be predictive of thesensitivity of a cancer to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione.

In one embodiment, IRF4/MUM1 expression in cancer cells, e.g.,ABC-subtype lymphoma, can be decreased with the treatment of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Insome embodiments, IRF4 downregulation by3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione canbe a potential pharmacodynamic biomarker.

In one embodiment, the cancer patients are multiple myeloma patients.

In one embodiment, the cancer patients are non-Hodgkin's lymphomapatients.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of DDB1 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of DDB2 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of IRF4 expression within the cancer.

In one embodiment, the method of selecting a group of cancer patients isbased on the level of NF-κB expression within the cancer.

In another embodiment, the method comprises selecting a group of cancerpatients responsive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof; based on the levelof CRBN expression, or the levels of DDB1, DDB2, IRF4 or NF-κBexpression within the patient's T cells, B cells, or plasma cells, forthe purposes of predicting clinical response, monitoring clinicalresponse, or monitoring patient compliance to dosing by3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, astereoisomer thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof.

In one embodiment, the cancer patients are selected from multiplemyeloma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, melanomaand solid tumor patients.

Also provided herein are methods of treating cancer, e.g., lymphoma,non-Hodgkin's lymphoma, multiple myeloma, leukemia, acute myeloidleukemia (AML), and solid tumors, which result in an improvement inoverall survival of the patient. In some embodiments, the improvement inoverall survival of the patient is observed in a patient populationsensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Insome embodiments, the patient population sensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione ischaracterized by one or more biomarkers provided herein.

In other embodiments, provided herein are methods of treating cancer,e.g., lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia,acute myeloid leukemia (AML), and solid tumors, which result in diseasefree survival of the patient. In some embodiments, disease free survivalof the patient is observed in a patient population sensitive totreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Insome embodiments, the patient population sensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione ischaracterized by one or more biomarkers provided herein.

In other embodiments, provided herein are methods of treating cancer,e.g., lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia,acute myeloid leukemia (AML), and solid tumors, which result in animprovement in the objective response rate in the patient population. Insome embodiments, the patient population sensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Insome embodiments, the patient population sensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione ischaracterized by one or more biomarkers provided herein.

In other embodiments, provided herein are methods of treating cancer,e.g., lymphoma, non-lymphoma, Hodgkin's lymphoma, multiple myeloma,leukemia, acute myeloid leukemia (AML), and solid tumors, which resultin an improved time to progression or progression-free survival of thepatient. In some embodiments, the improved time to progression orprogression-free survival of the patient is observed in a patientpopulation sensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Insome embodiments, the patient population sensitive to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione ischaracterized by one or more biomarkers provided herein.

Also provided herein are kits useful for predicting the likelihood of aneffective lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia,AML or solid tumor treatment or for monitoring the effectiveness of atreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. Thekit comprises a solid support, and a means for detecting the expressionof a biomarker in a biological sample. Such a kit may employ, forexample, a dipstick, a membrane, a chip, a disk, a test strip, a filter,a microsphere, a slide, a multiwell plate, or an optical fiber. Thesolid support of the kit can be, for example, a plastic, silicon, ametal, a resin, glass, a membrane, a particle, a precipitate, a gel, apolymer, a sheet, a sphere, a polysaccharide, a capillary, a film, aplate, or a slide. The biological sample can be, for example, a cellculture, a cell line, a tissue, an oral tissue, gastrointestinal tissue,an organ, an organelle, a biological fluid, a blood sample, a urinesample, or a skin sample. The biological sample can be, for example, alymph node biopsy, a bone marrow biopsy, or a sample of peripheral bloodtumor cells.

In one embodiment, the kit comprises a solid support, nucleic acidscontacting the support, where the nucleic acids are complementary to atleast 20, 50, 100, 200, 350, or more bases of mRNA of a gene associatedwith an activated B-cell phenotype in a NHL, and a means for detectingthe expression of the mRNA in a biological sample. In one embodiment,the gene associated with the activated B-cell phenotype is selected fromthe group consisting of IRF4/MUM1, FOXP1, SPIB, CARD11 and BLIMP/PDRM1.

In one embodiment, a kit useful for predicting the likelihood of aneffective lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia,AML or solid tumor treatment, or for monitoring the effectiveness of atreatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isprovided. The kit comprises a solid support, and a means for detectingthe expression of NF-κB in a biological sample. In one embodiment, thebiological sample is a cell culture or tissue sample. In one embodiment,the biological sample is a sample of tumor cells. In another embodiment,the biological sample is a lymph node biopsy, a bone marrow biopsy, or asample of peripheral blood tumor cells. In one embodiment, thebiological sample is a blood sample. In one embodiment, the NHL isDLBCL.

In certain embodiments, the kits provided herein employ means fordetecting the expression of a biomarker by quantitative real-time PCR(QT-PCR), microarray, flow cytometry or immunofluorescence. In otherembodiments, the expression of the biomarker is measured by ELISA-basedmethodologies or other similar methods known in the art. Additional mRNAand protein expression techniques may be used in connection with themethods and kits provided herein, e.g., CDNA hybridization andcytometric bead array methods.

In one embodiment, provided herein is a kit for predicting tumorresponse to treatment with3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione in anon-Hodgkin's lymphoma patient, comprising:

(i) a solid support; and(ii) a means for detecting the expression of a biomarker of an activatedB-cell phenotype of non-Hodgkin's lymphoma in a biological sample.

In one embodiment, the biomarker is NF-κB.

In one embodiment, the biomarker is a gene associated with the activatedB-cell phenotype and is selected from the group consisting of IRF4/MUM1,FOXP1, CARD11 and BLIMP/PDRM1.

In particular methods of the invention, a3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered in combination with a therapy conventionally used to treat,prevent or manage cancer. Examples of such conventional therapiesinclude, but are not limited to, surgery, chemotherapy, radiationtherapy, hormonal therapy, biological therapy and immunotherapy.

Also provided herein are pharmaceutical compositions, single unit dosageforms, dosing regimens and kits which comprise3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof, and a second, or additional, activeagent. Second active agents include specific combinations, or“cocktails,” of drugs.

In some embodiments, the methods for treating, preventing and/ormanaging lymphomas provided herein may be used in patients that have notresponded to standard treatment. In one embodiment, the lymphoma isrelapsed, refractory or resistant to conventional therapy.

In other embodiments, the methods for treating, preventing and/ormanaging lymphomas provided herein may be used in treatment naivepatients, i.e., patients that have not yet received treatment.

In certain embodiments,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, is administered in combination or alternation with atherapeutically effective amount of one or more additional activeagents. Second active agents include small molecules and large molecules(e.g., proteins and antibodies), examples of which are provided herein,as well as stem cells. Methods or therapies that can be used incombination with the administration of the compound provided hereininclude, but are not limited to, surgery, blood transfusions,immunotherapy, biological therapy, radiation therapy, and other non-drugbased therapies presently used to treat, prevent or manage disease andconditions associated with or characterized by undesired angiogenesis.

In one embodiment, the additional active agent is selected from thegroup consisting of an alkylating agent, an adenosine analog, aglucocorticoid, a kinase inhibitor, a SYK inhibitor, a PDE3 inhibitor, aPDE7 inhibitor, doxorubicin, chlorambucil, vincristine, bendamustine,forskolin, rituximab, or a combination thereof.

In one embodiment, the additional active agent is rituximab.

In one embodiment, the glucocorticoid is hydrocortisone ordexamethasone.

In one embodiment,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered in an amount of about 5 to about 50 mg per day.

In one embodiment,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered in an amount of about 5 to about 25 mg per day.

In another embodiment,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered in an amount of about 5, 10, 15, 25, 30 or 50 mg per day.

In another embodiment, 10 or 25 mg of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered per day.

In one embodiment,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered twice per day.

Provided herein are pharmaceutical compositions (e.g., single unitdosage forms) that can be used in methods disclosed herein. In certainembodiments, the pharmaceutical compositions comprise3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, and a second active agent.

In one embodiment,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isorally administered.

In one embodiment,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered in a capsule or tablet.

In one embodiment,3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione isadministered for 21 days followed by seven days rest in a 28 day cycle.

5.1 Definitions

To facilitate understanding of the disclosure set forth herein, a numberof terms are defined below.

The term “subject” or “patient” refers to an animal, including, but notlimited to, a mammal, including a primate (e.g., human), cow, sheep,goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and“patient” are used interchangeably herein in reference, for example, toa mammalian subject, such as a human subject.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” refer to the eradication or amelioration of adisease or disorder, or of one or more symptoms associated with thedisease or disorder. In certain embodiments, the terms refer tominimizing the spread or worsening of the disease or disorder resultingfrom the administration of one or more prophylactic or therapeuticagents to a patient with such a disease or disorder. In someembodiments, the terms refer to the administration of a compoundprovided herein, with or without other additional active agent, afterthe onset of symptoms of the particular disease.

As used herein, and unless otherwise specified, the terms “prevent,”“preventing” and “prevention” refer to the prevention of the onset,recurrence or spread of a disease or disorder, or of one or moresymptoms thereof. In certain embodiments, the terms refer to thetreatment with or administration of a compound provided herein, with orwithout other additional active compound, prior to the onset ofsymptoms, particularly to patients at risk of diseases or disordersprovided herein. The terms encompass the inhibition or reduction of asymptom of the particular disease. Patients with familial history of adisease in particular are candidates for preventive regimens in certainembodiments. In addition, patients who have a history of recurringsymptoms are also potential candidates for the prevention. In thisregard, the term “prevention” may be interchangeably used with the term“prophylactic treatment.”

As used herein, and unless otherwise specified, the terms “manage,”“managing” and “management” refer to preventing or slowing theprogression, spread or worsening of a disease or disorder, or of one ormore symptoms thereof. Often, the beneficial effects that a patientderives from a prophylactic and/or therapeutic agent do not result in acure of the disease or disorder. In this regard, the term “managing”encompasses treating a patient who had suffered from the particulardisease in an attempt to prevent or minimize the recurrence of thedisease, or lengthening the time during which the remains in remission.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment or management of a disease ordisorder, or to delay or minimize one or more symptoms associated withthe disease or disorder. A therapeutically effective amount of acompound means an amount of therapeutic agent, alone or in combinationwith other therapies, which provides a therapeutic benefit in thetreatment or management of the disease or disorder. The term“therapeutically effective amount” can encompass an amount that improvesoverall therapy, reduces or avoids symptoms or causes of disease ordisorder, or enhances the therapeutic efficacy of another therapeuticagent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent adisease or disorder, or prevent its recurrence. A prophylacticallyeffective amount of a compound means an amount of therapeutic agent,alone or in combination with other agents, which provides a prophylacticbenefit in the prevention of the disease. The term “prophylacticallyeffective amount” can encompass an amount that improves overallprophylaxis or enhances the prophylactic efficacy of anotherprophylactic agent.

The term “pharmaceutically acceptable carrier,” “pharmaceuticallyacceptable excipient,” “physiologically acceptable carrier,” or“physiologically acceptable excipient” refers to apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent, or encapsulatingmaterial. In one embodiment, each component is “pharmaceuticallyacceptable” in the sense of being compatible with the other ingredientsof a pharmaceutical formulation, and suitable for use in contact withthe tissue or organ of humans and animals without excessive toxicity,irritation, allergic response, immunogenicity, or other problems orcomplications, commensurate with a reasonable benefit/risk ratio. See,Remington: The Science and Practice of Pharmacy, 21st Edition;Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook ofPharmaceutical Excipients, 5th Edition; Rowe et al., Eds., ThePharmaceutical Press and the American Pharmaceutical Association: 2005;and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds.,Gower Publishing Company: 2007; Pharmaceutical Preformulation andFormulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004).

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. “Neoplastic,” as used herein, refers to anyform of dysregulated or unregulated cell growth, whether malignant orbenign, resulting in abnormal tissue growth. Thus, “neoplastic cells”include malignant and benign cells having dysregulated or unregulatedcell growth.

The term “relapsed” refers to a situation where a subject or a mammal,which has had a remission of cancer after therapy has a return of cancercells.

As used herein, an “effective patient tumor response” refers to anyincrease in the therapeutic benefit to the patient. An “effectivepatient tumor response” can be, for example, a 5%, 10%, 25%, 50%, or100% decrease in the rate of progress of the tumor. An “effectivepatient tumor response” can be, for example, a 5%, 10%, 25%, 50%, or100% decrease in the physical symptoms of a cancer. An “effectivepatient tumor response” can also be, for example, a 5%, 10%, 25%, 50%,100%, 200%, or more increase in the response of the patient, as measuredby any suitable means, such as gene expression, cell counts, assayresults, etc.

The term “likelihood” generally refers to an increase in the probabilityof an event. The term “likelihood” when used in reference to theeffectiveness of a patient tumor response generally contemplates anincreased probability that the rate of tumor progress or tumor cellgrowth will decrease. The term “likelihood” when used in reference tothe effectiveness of a patient tumor response can also generally meanthe increase of indicators, such as mRNA or protein expression, that mayevidence an increase in the progress in treating the tumor.

The term “predict” generally means to determine or tell in advance. Whenused to “predict” the effectiveness of a cancer treatment, for example,the term “predict” can mean that the likelihood of the outcome of thecancer treatment can be determined at the outset, before the treatmenthas begun, or before the treatment period has progressed substantially.

The term “monitor,” as used herein, generally refers to the overseeing,supervision, regulation, watching, tracking, or surveillance of anactivity. For example, the term “monitoring the effectiveness of acompound” refers to tracking the effectiveness in treating a cancer in apatient or in a tumor cell culture. Similarly, the “monitoring,” whenused in connection with patient compliance, either individually, or in aclinical trial, refers to the tracking or confirming that the patient isactually taking the immunomodulatory compound being tested asprescribed. The monitoring can be performed, for example, by followingthe expression of mRNA or protein biomarkers.

An improvement in the cancer or cancer-related disease can becharacterized as a complete or partial response. “Complete response”refers to an absence of clinically detectable disease with normalizationof any previously abnormal radiographic studies, bone marrow, andcerebrospinal fluid (CSF) or abnormal monoclonal protein measurements.“Partial response” refers to at least about a 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% decrease in all measurable tumor burden (i.e., thenumber of malignant cells present in the subject, or the measured bulkof tumor masses or the quantity of abnormal monoclonal protein) in theabsence of new lesions. The term “treatment” contemplates both acomplete and a partial response.

The term “refractory or resistant” refers to a circumstance where asubject or a mammal, even after intensive treatment, has residual cancercells in his body.

The term “drug resistance” refers to the condition when a disease doesnot respond to the treatment of a drug or drugs. Drug resistance can beeither intrinsic, which means the disease has never been responsive tothe drug or drugs, or it can be acquired, which means the disease ceasesresponding to a drug or drugs that the disease had previously respondedto. In certain embodiments, drug resistance is intrinsic. In certainembodiments, the drug resistance is acquired.

The term “sensitivity” and “sensitive” when made in reference totreatment with compound is a relative term which refers to the degree ofeffectiveness of the compound in lessening or decreasing the progress ofa tumor or the disease being treated. For example, the term “increasedsensitivity” when used in reference to treatment of a cell or tumor inconnection with a compound refers to an increase of, at least a 5%, ormore, in the effectiveness of the tumor treatment.

The term “expressed” or “expression” as used herein refers to thetranscription from a gene to give an RNA nucleic acid molecule at leastcomplementary in part to a region of one of the two nucleic acid strandsof the gene. The term “expressed” or “expression” as used herein alsorefers to the translation from the RNA molecule to give a protein, apolypeptide or a portion thereof.

An mRNA that is “upregulated” is generally increased upon a giventreatment or condition. An mRNA that is “downregulated” generally refersto a decrease in the level of expression of the mRNA in response to agiven treatment or condition. In some situations, the mRNA level canremain unchanged upon a given treatment or condition.

An mRNA from a patient sample can be “upregulated” when treated with animmunomodulatory compound, as compared to a non-treated control. Thisupregulation can be, for example, an increase of about 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1,000%, 5,000% ormore of the comparative control mRNA level.

Alternatively, an mRNA can be “downregulated”, or expressed at a lowerlevel, in response to administration of certain immunomodulatorycompounds or other agents. A downregulated mRNA can be, for example,present at a level of about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, 10%, 1% or less of the comparative control mRNA level.

Similarly, the level of a polypeptide or protein biomarker from apatient sample can be increased when treated with an immunomodulatorycompound, as compared to a non-treated control. This increase can beabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%,500%, 1,000%, 5,000% or more of the comparative control protein level.

Alternatively, the level of a protein biomarker can be decreased inresponse to administration of certain immunomodulatory compounds orother agents. This decrease can be, for example, present at a level ofabout 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1% or lessof the comparative control protein level.

The terms “determining”, “measuring”, “evaluating”, “assessing” and“assaying” as used herein generally refer to any form of measurement,and include determining if an element is present or not. These termsinclude both quantitative and/or qualitative determinations. Assessingmay be relative or absolute. “Assessing the presence of” can includedetermining the amount of something present, as well as determiningwhether it is present or absent.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases know in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

As used herein and unless otherwise indicated, the term “solvate” meansa compound provided herein or a salt thereof, that further includes astoichiometric or non-stoichiometric amount of solvent bound bynon-covalent intermolecular forces. Where the solvent is water, thesolvate is a hydrate.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,derivatives of the compound of Formula I provided herein that comprisebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Otherexamples of prodrugs include derivatives of the compound of Formula Iprovided herein that comprise —NO, —NO₂, —ONO, or —ONO₂ moieties.Prodrugs can be prepared using such methods as described in Burger'sMedicinal Chemistry and Drug Discovery, 172-178, 949-982 (Manfred E.Wolff ed., 5th ed. 1995), and Design of Prodrugs (H. Bundgaard ed.,Elselvier, New York 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,” and“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyl-oxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, amino acids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

As used herein and unless otherwise indicated, the term “stereomericallypure” means a composition that comprises one stereoisomer of a compoundand is substantially free of other stereoisomers of that compound. Forexample, a stereomerically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. A stereomerically pure composition of a compound havingtwo chiral centers will be substantially free of other diastereomers ofthe compound. In certain embodiments, a stereomerically pure compoundcomprises greater than about 80% by weight of one stereoisomer of thecompound and less than about 20% by weight of other stereoisomers of thecompound, greater than about 90% by weight of one stereoisomer of thecompound and less than about 10% by weight of the other stereoisomers ofthe compound, greater than about 95% by weight of one stereoisomer ofthe compound and less than about 5% by weight of the other stereoisomersof the compound, or greater than about 97% by weight of one stereoisomerof the compound and less than about 3% by weight of the otherstereoisomers of the compound. As used herein and unless otherwiseindicated, the term “stereomerically enriched” means a composition thatcomprises greater than about 60% by weight of one stereoisomer of acompound, greater than about 70% by weight, or greater than about 80% byweight of one stereoisomer of a compound. As used herein and unlessotherwise indicated, the term “enantiomerically pure” means astereomerically pure composition of a compound having one chiral center.Similarly, the term “stereomerically enriched” means a stereomericallyenriched composition of a compound having one chiral center.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

5.2 Clinical Trials Endpoints for Cancer Approval

“Overall survival” is defined as the time from randomization until deathfrom any cause, and is measured in the intent-to-treat population.Overall survival should be evaluated in randomized controlled studies.Demonstration of a statistically significant improvement in overallsurvival can be considered to be clinically significant if the toxicityprofile is acceptable, and has often supported new drug approval.

Several endpoints are based on tumor assessments. These endpointsinclude disease free survival (DFS), objective response rate (ORR), timeto progression (TTP), progression-free survival (PFS), andtime-to-treatment failure (TTF). The collection and analysis of data onthese time-dependent endpoints are based on indirect assessments,calculations, and estimates (e.g., tumor measurements).

Generally, “disease free survival” (DFS) is defined as the time fromrandomization until recurrence of tumor or death from any cause.Although overall survival is a conventional endpoint for most adjuvantsettings, DFS can be an important endpoint in situations where survivalmay be prolonged, making a survival endpoint impractical. DFS can be asurrogate for clinical benefit or it can provide direct evidence ofclinical benefit. This determination is based on the magnitude of theeffect, its risk-benefit relationship, and the disease setting. Thedefinition of DFS can be complicated, particularly when deaths are notedwithout prior tumor progression documentation. These events can bescored either as disease recurrences or as censored events. Although allmethods for statistical analysis of deaths have some limitations,considering all deaths (deaths from all causes) as recurrences canminimize bias. DFS can be overestimated using this definition,especially in patients who die after a long period without observation.Bias can be introduced if the frequency of long-term follow-up visits isdissimilar between the study arms or if dropouts are not random becauseof toxicity.

“Objective response rate” (ORR) is defined as the proportion of patientswith tumor size reduction of a predefined amount and for a minimum timeperiod. Response duration usually is measured from the time of initialresponse until documented tumor progression. Generally, the FDA hasdefined ORR as the sum of partial responses plus complete responses.When defined in this manner, ORR is a direct measure of drug antitumoractivity, which can be evaluated in a single-arm study. If available,standardized criteria should be used to ascertain response. A variety ofresponse criteria have been considered appropriate (e.g., RECISTcriteria) (Therasse et al., (2000) J. Natl. Cancer Inst, 92: 205-16).The significance of ORR is assessed by its magnitude and duration, andthe percentage of complete responses (no detectable evidence of tumor).

“Time to progression” (TTP) and “progression-free survival” (PFS) haveserved as primary endpoints for drug approval. TTP is defined as thetime from randomization until objective tumor progression; TTP does notinclude deaths. PFS is defined as the time from randomization untilobjective tumor progression or death. Compared with TTP, PFS is thepreferred regulatory endpoint. PFS includes deaths and thus can be abetter correlate to overall survival. PFS assumes patient deaths arerandomly related to tumor progression. However, in situations where themajority of deaths are unrelated to cancer, TTP can be an acceptableendpoint.

As an endpoint to support drug approval, PFS can reflect tumor growthand be assessed before the determination of a survival benefit. Itsdetermination is not confounded by subsequent therapy. For a givensample size, the magnitude of effect on PFS can be larger than theeffect on overall survival. However, the formal validation of PFS as asurrogate for survival for the many different malignancies that existcan be difficult. Data are sometimes insufficient to allow a robustevaluation of the correlation between effects on survival and PFS.Cancer trials are often small, and proven survival benefits of existingdrugs are generally modest. The role of PFS as an endpoint to supportlicensing approval varies in different cancer settings. Whether animprovement in PFS represents a direct clinical benefit or a surrogatefor clinical benefit depends on the magnitude of the effect and therisk-benefit of the new treatment compared to available therapies.

“Time-to-treatment failure” (TTF) is defined as a composite endpointmeasuring time from randomization to discontinuation of treatment forany reason, including disease progression, treatment toxicity, anddeath. TTF is not recommended as a regulatory endpoint for drugapproval. TTF does not adequately distinguish efficacy from theseadditional variables. A regulatory endpoint should clearly distinguishthe efficacy of the drug from toxicity, patient or physician withdrawal,or patient intolerance.

5.3 the Compound

The compound suitable for use in the methods provided herein is3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,having the structure of Formula I:

or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof.

The compound of Formula I can be prepared according to the methodsdescribed in the Examples provided herein or as described in U.S. Pat.No. 7,635,700, the disclosure of which is incorporated herein byreference in its entirety. The compound can be also synthesizedaccording to other methods apparent to those of skill in the art basedupon the teaching herein.

The compound of Formula I markedly inhibits TNF-α, IL-1β, and otherinflammatory cytokines in LPS-stimulated hPBMC and human whole blood.TNF-α is an inflammatory cytokine produced by macrophages and monocytesduring acute inflammation. TNF-α is responsible for a diverse range ofsignaling events within cells. TNF-α may play a pathological role incancer. Without being limited by theory, one of the biological effectsexerted by the immunomodulatory compound of Formula I is the reductionof synthesis of TNF-α. The immunomodulatory compound of Formula Ienhances the degradation of TNF-α mRNA. The compound of Formula I alsopotently inhibits IL-1β and stimulates IL-10 under these conditions.

Further, without being limited by theory, the compound of Formula I is apotent co-stimulator of T cells and increase cell proliferation in adose dependent manner under appropriate conditions.

In certain embodiments, without being limited by theory, the biologicaleffects exerted by the immunomodulatory compound of Formula I include,but not limited to, anti-angiogenic and immune modulating effects.

In certain embodiments, the compound of Formula I is a solid. In certainembodiments, the compound of Formula I is hydrated. In certainembodiments, the compound of Formula I is solvated. In certainembodiments, the compound of Formula I is anhydrous. In certainembodiments, the compound of Formula I is nonhygroscopic.

In certain embodiments, the solid compound of Formula I is amorphous. Incertain embodiments, the solid compound of Formula I is crystalline. Incertain embodiments, the solid compound of Formula I is in a crystallineform described in U.S. Provisional Pat. App. No. 61/451,806, filed Mar.11, 2011, which is incorporated herein by reference in its entirety.

The solid forms of the compound of Formula I can be prepared accordingto the methods described in the disclosure of U.S. Provisional Pat. App.No. 61/451,806. The solid forms can be also prepared according to othermethods apparent to those of skill in the art.

In certain embodiments, the compound of Formula I is a hydrochloridesalt of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable solvate, hydrate, co-crystal, clathrate, or polymorphthereof. In certain embodiments, the hydrochloride salt is a solid. Incertain embodiments, the hydrochloride salt is anhydrous. In certainembodiments, the hydrochloride salt is nonhygroscopic. In certainembodiments, the hydrochloride salt is amorphous. In certainembodiments, the hydrochloride salt is crystalline. In certainembodiments, the hydrochloride salt is in crystalline Form A.

The hydrochloride salt of the compound of Formula I and solid formsthereof can be prepared according to the methods described in thedisclosure of U.S. Provisional Pat. App. No. 61/451,806. Thehydrochloride salt the solid forms thereof can be also preparedaccording to other methods apparent to those of skill in the art.

The compound of Formula I provided herein contains one chiral center,and can exist as a mixture of enantiomers, e.g., a racemic mixture. Thisdisclosure encompasses the use of stereomerically pure forms of such acompound, as well as the use of mixtures of those forms. For example,mixtures comprising equal or unequal amounts of the enantiomers of thecompound of Formula I provided herein may be used in methods andcompositions disclosed herein. These isomers may be asymmetricallysynthesized or resolved using standard techniques such as chiral columnsor chiral resolving agents. See, e.g., Jacques, J., et al., Enantiomers,Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen,S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, S. H., Tablesof Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed.,Univ. of Notre Dame Press, Notre Dame, Ind., 1972).

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of the structure.

5.4 Second Active Agents

A compound provided herein, e.g., the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, can be combined with one or more other pharmacologically activecompounds (“second active agents”) in methods and compositions providedherein. It is believed that certain combinations work synergistically inthe treatment of particular types of cancer, and certain diseases andconditions associated with or characterized by undesired angiogenesis.The compound of Formula I provided herein can also work to alleviateadverse effects associated with certain second active agents, and somesecond active agents can be used to alleviate adverse effects associatedwith the compound of Formula I provided herein.

One or more second active ingredients or agents can be used in themethods and compositions provided herein with the compound of Formula Iprovided herein. Second active agents can be large molecules (e.g.,proteins) or small molecules (e.g., synthetic inorganic, organometallic,or organic molecules).

Examples of large molecule active agents include, but are not limitedto, hematopoietic growth factors, cytokines, and monoclonal andpolyclonal antibodies. In certain embodiments, large molecule activeagents are biological molecules, such as naturally occurring orartificially made proteins. Proteins that are particularly useful inthis disclosure include proteins that stimulate the survival and/orproliferation of hematopoietic precursor cells and immunologicallyactive poietic cells in vitro or in vivo. Others stimulate the divisionand differentiation of committed erythroid progenitors in cells in vitroor in vivo. Particular proteins include, but are not limited to:interleukins, such as IL-2 (including recombinant IL-II (“rIL2”) andcanarypox IL-2), IL-10, IL-12, and IL-18; interferons, such asinterferon alfa-2a, interferon alfa-2b, interferon alfa-n1, interferonalfa-n3, interferon beta-I a, and interferon gamma-I b; GM-CF andGM-CSF; and EPO.

Particular proteins that can be used in the methods and compositions ofthe disclosure include, but are not limited to: filgrastim, which issold in the United States under the trade name NEUPOGEN® (Amgen,Thousand Oaks, Calif.); sargramostim, which is sold in the United Statesunder the trade name LEUKINE® (Immunex, Seattle, Wash.); and recombinantEPO, which is sold in the United States under the trade name EPGEN®(Amgen, Thousand Oaks, Calif.).

Inhibitors of ActRII receptors or activin-ActRII inhibitors may be usedin the methods and compositions provided herein. ActRII receptorsinclude ActRIIA inhibitors and ActRIIB inhibitors. Inhibitors of ActRIIreceptors can be polypeptides comprising activin-binding domains ofActRII. In certain embodiments, the activin-binding domain comprisingpolypeptides are linked to an Fc portion of an antibody (i.e., aconjugate comprising an activin-binding domain comprising polypeptide ofan ActRII receptor and an Fc portion of an antibody is generated). Incertain embodiments, the activin-binding domain is linked to an Fcportion of an antibody via a linker, e.g., a peptide linker. Examples ofsuch non-antibody proteins selected for activin or ActRIIA binding andmethods for design and selection of the same are found inWO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989, US2003/0133939, and US 2005/0238646, each of which is incorporated hereinby reference in its entirety.

Recombinant and mutated forms of GM-CSF can be prepared as described inU.S. Pat. Nos. 5,391,485; 5,393,870; and 5,229,496; the disclosure ofeach of which is incorporated herein by reference in its entirety.Recombinant and mutated forms of G-CSF can be prepared as described inU.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755; thedisclosure of each of which is incorporated herein by reference in itsentirety.

This disclosure encompasses the use of native, naturally occurring, andrecombinant proteins. The disclosure further encompasses mutants andderivatives (e.g., modified forms) of naturally occurring proteins thatexhibit, in vivo, at least some of the pharmacological activity of theproteins upon which they are based. Examples of mutants include, but arenot limited to, proteins that have one or more amino acid residues thatdiffer from the corresponding residues in the naturally occurring formsof the proteins. Also encompassed by the term “mutants” are proteinsthat lack carbohydrate moieties normally present in their naturallyoccurring forms (e.g., nonglycosylated forms). Examples of derivativesinclude, but are not limited to, pegylated derivatives and fusionproteins, such as proteins formed by fusing IgG1 or IgG3 to the proteinor active portion of the protein of interest. See, e.g., Penichet, M. L.and Morrison, S. L., J. Immunol. Methods 248:91-101 (2001).

Antibodies that can be used in combination with the compound of FormulaI provided herein include monoclonal and polyclonal antibodies. Examplesof antibodies include, but are not limited to, trastuzumab (HERCEPTIN®),rituximab (RITUXAN®), bevacizumab (AVASTIN™), pertuzumab (OMNITARG™),tositumomab (BEXXAR®), edrecolomab (PANOREX®), panitumumab and G250. Thecompound of Formula I provided herein can also be combined with or usedin combination with anti-TNF-α antibodies.

Large molecule active agents may be administered in the form ofanti-cancer vaccines. For example, vaccines that secrete, or cause thesecretion of, cytokines such as IL-2, SCF, CXCl4 (platelet factor 4),G-CSF, and GM-CSF can be used in the methods, pharmaceuticalcompositions, and kits of the disclosure. See, e.g., Emens, L. A., etal., Curr. Opinion Mol. Ther. 3(1):77-84 (2001).

Second active agents that are small molecules can also be used toalleviate adverse effects associated with the administration of thecompound of Formula I provided herein. However, like some largemolecules, many are believed to be capable of providing a synergisticeffect when administered with (e.g., before, after or simultaneously)the compound of Formula I. Examples of small molecule second activeagents include, but are not limited to, anti-cancer agents, antibiotics,immunosuppressive agents, and steroids.

Examples of anti-cancer agents include, but are not limited to:abraxane; ace-11; acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantroneacetate; amrubicin; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib(COX-2 inhibitor); chlorambucil; cirolemycin; cisplatin; cladribine;crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine;dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin;dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin;doxorubicin hydrochloride; droloxifene; droloxifene citrate;dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; herceptin; hydroxyurea; idarubicin hydrochloride;ifosfamide; ilmofosine; iproplatin; irinotecan; irinotecanhydrochloride; lanreotide acetate; lapatinib; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; romidepsin; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; stem cell treatments such asPDA-001; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalansodium; taxotere; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride.

Other anti-cancer drugs include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;b-FGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC®),imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemiainhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide;mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; Erbitux, human chorionic gonadotrophin; monophosphoryllipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; oblimersen (GENASENSE®);O⁶-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine;romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin;SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine;senescence derived inhibitor 1; sense oligonucleotides; signaltransduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate;sodium phenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; translation inhibitors; tretinoin;triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron;turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;ubenimex; urogenital sinus-derived growth inhibitory factor; urokinasereceptor antagonists; vapreotide; variolin B; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

Specific second active agents include, but are not limited to,oblimersen) (GENASENSE®, remicade, docetaxel, celecoxib, melphalan,dexamethasone (DECADRON®), steroids, gemcitabine, cisplatinum,temozolomide, etoposide, cyclophosphamide, temodar, carboplatin,procarbazine, gliadel, tamoxifen, topotecan, methotrexate, ARISA®,taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11,interferon alpha, pegylated interferon alpha (e.g., PEG INTRON-A),capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomaldaunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2,GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin,busulphan, prednisone, bisphosphonate, arsenic trioxide, vincristine,doxorubicin (DOXIL®), paclitaxel, ganciclovir, adriamycin, estramustinesodium phosphate (EMCYT®), sulindac, and etoposide.

5.5 Biomarkers

Provided herein are methods relating to the use of mRNAs or proteins asbiomarkers to ascertain the effectiveness of cancer therapy. mRNA orprotein levels can be used to determine whether a particular agent islikely to be successful in the treatment of a specific type of cancer,e.g., non-Hodgkin's lymphoma.

A biological marker or “biomarker” is a substance whose detectionindicates a particular biological state, such as, for example, thepresence of cancer. In some embodiments, biomarkers can either bedetermined individually, or several biomarkers can be measuredsimultaneously.

In some embodiments, a “biomarker” indicates a change in the level ofmRNA expression that may correlate with the risk or progression of adisease, or with the susceptibility of the disease to a given treatment.In some embodiments, the biomarker is a nucleic acid, such as a mRNA orcDNA.

In additional embodiments, a “biomarker” indicates a change in the levelof polypeptide or protein expression that may correlate with the risk,susceptibility to treatment, or progression of a disease. In someembodiments, the biomarker can be a polypeptide or protein, or afragment thereof. The relative level of specific proteins can bedetermined by methods known in the art. For example, antibody basedmethods, such as an immunoblot, enzyme-linked immunosorbent assay(ELISA), or other methods can be used.

5.6 Methods of Treatment and Prevention

In one embodiment, provided herein is a method of treating andpreventing cancer, which comprises administering to a patient a compoundprovided herein, e.g., the compound of Formula I, or an enantiomer or amixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

In another embodiment, provided herein is method of managing cancer,which comprises administering to a patient a compound provided herein,e.g., the compound of Formula I, or an enantiomer or a mixture ofenantiomers thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof. Provided hereinare methods of treating or managing lymphoma, particularly non-Hodgkin'slymphoma. In some embodiments, provided herein are methods for thetreatment or management of non-Hodgkin's lymphoma (NHL), including butnot limited to, diffuse large B-cell lymphoma (DLBCL), using prognosticfactors.

Also provided herein are methods of treating patients who have beenpreviously treated for cancer but are non-responsive to standardtherapies, as well as those who have not previously been treated. Theinvention also encompasses methods of treating patients regardless ofpatient's age, although some diseases or disorders are more common incertain age groups. The invention further encompasses methods oftreating patients who have undergone surgery in an attempt to treat thedisease or condition at issue, as well as those who have not. Becausepatients with cancer have heterogeneous clinical manifestations andvarying clinical outcomes, the treatment given to a patient may vary,depending on his/her prognosis. The skilled clinician will be able toreadily determine without undue experimentation specific secondaryagents, types of surgery, and types of non-drug based standard therapythat can be effectively used to treat an individual patient with cancer.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood born tumors. The term “cancer” refers to disease ofskin tissues, organs, blood, and vessels, including, but not limited to,cancers of the bladder, bone, blood, brain, breast, cervix, chest,colon, endrometrium, esophagus, eye, head, kidney, liver, lymph nodes,lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis,throat, and uterus. Specific cancers include, but are not limited to,advanced malignancy, amyloidosis, neuroblastoma, meningioma,hemangiopericytoma, multiple brain metastase, glioblastoma multiforms,glioblastoma, brain stem glioma, poor prognosis malignant brain tumor,malignant glioma, recurrent malignant giolma, anaplastic astrocytoma,anaplastic oligodendroglioma, neuroendocrine tumor, rectaladenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectalcarcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma,karotype acute myeloblastic leukemia, Hodgkin's lymphoma, non-Hodgkin'slymphoma, cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuselarge B-Cell lymphoma, low grade follicular lymphoma, malignantmelanoma, malignant mesothelioma, malignant pleural effusionmesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma,gynecologic sarcoma, soft tissue sarcoma, scleroderma, cutaneousvasculitis, Langerhans cell histiocytosis, leiomyosarcoma,fibrodysplasia ossificans progressive, hormone refractory prostatecancer, resected high-risk soft tissue sarcoma, unrescectablehepatocellular carcinoma, Waldenstrom's macroglobulinemia, smolderingmyeloma, indolent myeloma, fallopian tube cancer, androgen independentprostate cancer, androgen dependent stage IV non-metastatic prostatecancer, hormone-insensitive prostate cancer, chemotherapy-insensitiveprostate cancer, papillary thyroid carcinoma, follicular thyroidcarcinoma, medullary thyroid carcinoma, and leiomyoma

In certain embodiments, the cancer is a blood borne tumor. In certainembodiments, the blood borne tumor is metastatic. In certainembodiments, the blood borne tumor is drug resistant. In certainembodiments, the cancer is myeloma or lymphoma.

In certain embodiments, the cancer is a solid tumor. In certainembodiments, the solid tumor is metastatic. In certain embodiments, thesolid tumor is drug-resistant. In certain embodiments, the solid tumoris hepatocellular carcinoma, prostate cancer, ovarian cancer, orglioblastoma.

In certain embodiments, a therapeutically or prophylactically effectiveamount of the compound is from about 0.005 to about 1,000 mg per day,from about 0.01 to about 500 mg per day, from about 0.01 to about 250 mgper day, from about 0.01 to about 100 mg per day, from about 0.1 toabout 100 mg per day, from about 0.5 to about 100 mg per day, from about1 to about 100 mg per day, from about 0.01 to about 50 mg per day, fromabout 0.1 to about 50 mg per day, from about 0.5 to about 50 mg per day,from about 1 to about 50 mg per day, from about 0.02 to about 25 mg perday, or from about 0.05 to about 10 mg per day.

In certain embodiment, a therapeutically or prophylactically effectiveamount is from about 0.005 to about 1,000 mg per day, from about 0.01 toabout 500 mg per day, from about 0.01 to about 250 mg per day, fromabout 0.01 to about 100 mg per day, from about 0.1 to about 100 mg perday, from about 0.5 to about 100 mg per day, from about 1 to about 100mg per day, from about 0.01 to about 50 mg per day, from about 0.1 toabout 50 mg per day, from about 0.5 to about 50 mg per day, from about 1to about 50 mg per day, from about 0.02 to about 25 mg per day, or fromabout 0.05 to about 10 mg every other day.

In certain embodiments, the therapeutically or prophylacticallyeffective amount is about 1, about 2, about 5, about 10, about 15, about20, about 25, about 30, about 40, about 45, about 50, about 60, about70, about 80, about 90, about 100, or about 150 mg per day.

In one embodiment, the recommended daily dose range of the compound ofFormula I for the conditions described herein lie within the range offrom about 0.5 mg to about 50 mg per day, preferably given as a singleonce-a-day dose, or in divided doses throughout a day. In someembodiments, the dosage ranges from about 1 mg to about 50 mg per day.In other embodiments, the dosage ranges from about 0.5 to about 5 mg perday. Specific doses per day include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49 or 50 mg per day.

In a specific embodiment, the recommended starting dosage may be 0.5, 1,2, 3, 4, 5, 10, 15, 20, 25 or 50 mg per day. In another embodiment, therecommended starting dosage may be 0.5, 1, 2, 3, 4, or 5 mg per day. Thedose may be escalated to 15, 20, 25, 30, 35, 40, 45 and 50 mg/day. In aspecific embodiment, the compound can be administered in an amount ofabout 25 mg/day to patients with NHL (e.g., DLBCL). In a particularembodiment, the compound can be administered in an amount of about 10mg/day to patients with NHL (e.g., DLBCL).

In certain embodiments, the therapeutically or prophylacticallyeffective amount is from about 0.001 to about 100 mg/kg/day, from about0.01 to about 50 mg/kg/day, from about 0.01 to about 25 mg/kg/day, fromabout 0.01 to about 10 mg/kg/day, from about 0.01 to about 9 mg/kg/day,0.01 to about 8 mg/kg/day, from about 0.01 to about 7 mg/kg/day, fromabout 0.01 to about 6 mg/kg/day, from about 0.01 to about 5 mg/kg/day,from about 0.01 to about 4 mg/kg/day, from about 0.01 to about 3mg/kg/day, from about 0.01 to about 2 mg/kg/day, or from about 0.01 toabout 1 mg/kg/day.

The administered dose can also be expressed in units other thanmg/kg/day. For example, doses for parenteral administration can beexpressed as mg/m²/day. One of ordinary skill in the art would readilyknow how to convert doses from mg/kg/day to mg/m²/day to given eitherthe height or weight of a subject or both (see,www.fda.gov/cder/cancer/animalframe.htm). For example, a dose of 1mg/kg/day for a 65 kg human is approximately equal to 38 mg/m²/day.

In certain embodiments, the amount of the compound administered issufficient to provide a plasma concentration of the compound at steadystate, ranging from about 0.001 to about 500 μM, about 0.002 to about200 μM, about 0.005 to about 100 μM, about 0.01 to about 50 μM, fromabout 1 to about 50 μM, about 0.02 to about 25 μM, from about 0.05 toabout 20 μM, from about 0.1 to about 20 μM, from about 0.5 to about 20μM, or from about 1 to about 20 μM.

In other embodiments, the amount of the compound administered issufficient to provide a plasma concentration of the compound at steadystate, ranging from about 5 to about 100 nM, about 5 to about 50 nM,about 10 to about 100 nM, about 10 to about 50 nM or from about 50 toabout 100 nM.

As used herein, the term “plasma concentration at steady state” is theconcentration reached after a period of administration of a compoundprovided herein, e.g., the compound of Formula I, or an enantiomer or amixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof. Oncesteady state is reached, there are minor peaks and troughs on the timedependent curve of the plasma concentration of the compound.

In certain embodiments, the amount of the compound administered issufficient to provide a maximum plasma concentration (peakconcentration) of the compound, ranging from about 0.001 to about 500μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01to about 50 μM, from about 1 to about 50 μM, about 0.02 to about 25 μM,from about 0.05 to about 20 μM, from about 0.1 to about 20 μM, fromabout 0.5 to about 20 μM, or from about 1 to about 20 μM.

In certain embodiments, the amount of the compound administered issufficient to provide a minimum plasma concentration (troughconcentration) of the compound, ranging from about 0.001 to about 500μM, about 0.002 to about 200 μM, about 0.005 to about 100 μM, about 0.01to about 50 μM, from about 1 to about 50 μM, about 0.01 to about 25 μM,from about 0.01 to about 20 μM, from about 0.02 to about 20 μM, fromabout 0.02 to about 20 μM, or from about 0.01 to about 20 μM.

In certain embodiments, the amount of the compound administered issufficient to provide an area under the curve (AUC) of the compound,ranging from about 100 to about 100,000 ng*hr/mL, from about 1,000 toabout 50,000 ng*hr/mL, from about 5,000 to about 25,000 ng*hr/mL, orfrom about 5,000 to about 10,000 ng*hr/mL.

In certain embodiments, the patient to be treated with one of themethods provided herein has not been treated with anticancer therapyprior to the administration of the compound of Formula I. In certainembodiments, the patient to be treated with one of the methods providedherein has been treated with anticancer therapy prior to theadministration of the compound of Formula I. In certain embodiments, thepatient to be treated with one of the methods provided herein hasdeveloped drug resistance to the anticancer therapy.

The methods provided herein encompass treating a patient regardless ofpatient's age, although some diseases or disorders are more common incertain age groups. Further provided herein is a method for treating apatient who has undergone surgery in an attempt to treat the disease orcondition at issue, as well in one who has not. Because the subjectswith cancer have heterogeneous clinical manifestations and varyingclinical outcomes, the treatment given to a particular subject may vary,depending on his/her prognosis. The skilled clinician will be able toreadily determine without undue experimentation, specific secondaryagents, types of surgery, and types of non-drug based standard therapythat can be effectively used to treat an individual subject with cancer.

Depending on the disease to be treated and the subject's condition, thecompound of Formula I, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, may be administered byoral, parenteral (e.g., intramuscular, intraperitoneal, intravenous,CIV, intracistemal injection or infusion, subcutaneous injection, orimplant), inhalation, nasal, vaginal, rectal, sublingual, or topical(e.g., transdermal or local) routes of administration. The compound ofFormula I, or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, may be formulated, alone or together,in suitable dosage unit with pharmaceutically acceptable excipients,carriers, adjuvants and vehicles, appropriate for each route ofadministration.

In one embodiment, the compound of Formula I, or an enantiomer or amixture of enantiomers thereof; or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered orally. In another embodiment, the compound of Formula I,or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered parenterally. In yetanother embodiment, the compound of Formula I, or an enantiomer or amixture of enantiomers thereof; or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered intravenously.

The compound of Formula I, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, can be delivered as asingle dose such as, e.g., a single bolus injection, or oral tablets orpills; or over time, such as, e.g., continuous infusion over time ordivided bolus doses over time. The compound can be administeredrepeatedly if necessary, for example, until the patient experiencesstable disease or regression, or until the patient experiences diseaseprogression or unacceptable toxicity. For example, stable disease forsolid tumors generally means that the perpendicular diameter ofmeasurable lesions has not increased by 25% or more from the lastmeasurement. Response Evaluation Criteria in Solid Tumors (RECIST)Guidelines, Journal of the National Cancer Institute 92(3): 205-216(2000). Stable disease or lack thereof is determined by methods known inthe art such as evaluation of patient symptoms, physical examination,visualization of the tumor that has been imaged using X-ray, CAT, PET,or Mill scan and other commonly accepted evaluation modalities.

The compound of Formula I, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, can be administered oncedaily (QD), or divided into multiple daily doses such as twice daily(BID), three times daily (TID), and four times daily (QID). In addition,the administration can be continuous (i.e., daily for consecutive daysor every day), intermittent, e.g., in cycles (i.e., including days,weeks, or months of rest without drug). As used herein, the term “daily”is intended to mean that a therapeutic compound, such as the compound ofFormula I, is administered once or more than once each day, for example,for a period of time. The term “continuous” is intended to mean that atherapeutic compound, such as the compound of Formula I, is administereddaily for an uninterrupted period of at least 10 days to 52 weeks. Theterm “intermittent” or “intermittently” as used herein is intended tomean stopping and starting at either regular or irregular intervals. Forexample, intermittent administration of the compound of Formula I isadministration for one to six days per week, administration in cycles(e.g., daily administration for two to eight consecutive weeks, then arest period with no administration for up to one week), oradministration on alternate days. The term “cycling” as used herein isintended to mean that a therapeutic compound, such as the compound ofFormula I, is administered daily or continuously but with a rest period.

In some embodiments, the frequency of administration is in the range ofabout a daily dose to about a monthly dose. In certain embodiments,administration is once a day, twice a day, three times a day, four timesa day, once every other day, twice a week, once every week, once everytwo weeks, once every three weeks, or once every four weeks. In oneembodiment, the compound of Formula I, or an enantiomer or a mixture ofenantiomers thereof; or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof, is administeredonce a day. In another embodiment, the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, is administered twice a day. In yet another embodiment, thecompound of Formula I, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, is administered three timesa day. In still another embodiment, the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, is administered four times a day.

In certain embodiments, the compound of Formula I, or an enantiomer or amixture of enantiomers thereof; or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered once per day from one day to six months, from one week tothree months, from one week to four weeks, from one week to three weeks,or from one week to two weeks. In certain embodiments, the compound ofFormula I, or a pharmaceutically acceptable salt or solvate thereof, isadministered once per day for one week, two weeks, three weeks, or fourweeks. In one embodiment, the compound of Formula I, or an enantiomer ora mixture of enantiomers thereof; or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered once per day for one week. In another embodiment, thecompound of Formula I, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, is administered once perday for two weeks. In yet another embodiment, the compound of Formula I,or an enantiomer or a mixture of enantiomers thereof; or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered once per day for threeweeks. In still another embodiment, the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, is administered once per day for four weeks.

5.6.1 Combination Therapy with a Second Active Agent

The compound of Formula I, or an enantiomer or a mixture of enantiomersthereof; or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, can also be combined orused in combination with other therapeutic agents useful in thetreatment and/or prevention of cancer described herein.

In one embodiment, provided herein is a method of treating, preventing,or managing cancer, comprising administering to a patient3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof; in combination with one or more second active agents, andoptionally in combination with radiation therapy, blood transfusions, orsurgery. Examples of second active agents are disclosed herein (see,e.g., section 5.3).

As used herein, the term “in combination” includes the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).However, the use of the term “in combination” does not restrict theorder in which therapies (e.g., prophylactic and/or therapeutic agents)are administered to a patient with a disease or disorder. A firsttherapy (e.g., a prophylactic or therapeutic agent such as a compoundprovided herein, a compound provided herein, e.g., the compound ofFormula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof) can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapy (e.g., a prophylactic or therapeutic agent) to thesubject. Triple therapy is also contemplated herein.

Administration of the compound of Formula I and one or more secondactive agents to a patient can occur simultaneously or sequentially bythe same or different routes of administration. The suitability of aparticular route of administration employed for a particular activeagent will depend on the active agent itself (e.g., whether it can beadministered orally without decomposing prior to entering the bloodstream) and the cancer being treated.

The route of administration of the compound of Formula I is independentof the route of administration of a second therapy. In one embodiment,the compound of Formula I is administered orally. In another embodiment,the compound of Formula I is administered intravenously. Thus, inaccordance with these embodiments, the compound of Formula I isadministered orally or intravenously, and the second therapy can beadministered orally, parenterally, intraperitoneally, intravenously,intraarterially, transdermally, sublingually, intramuscularly, rectally,transbuccally, intranasally, liposomally, via inhalation, vaginally,intraoccularly, via local delivery by catheter or stent, subcutaneously,intraadiposally, intraarticularly, intrathecally, or in a slow releasedosage form. In one embodiment, the compound of Formula I and a secondtherapy are administered by the same mode of administration, orally orby IV. In another embodiment, the compound of Formula I is administeredby one mode of administration, e.g., by IV, whereas the second agent (ananticancer agent) is administered by another mode of administration,e.g., orally.

In one embodiment, the second active agent is administered intravenouslyor subcutaneously and once or twice daily in an amount of from about 1to about 1000 mg, from about 5 to about 500 mg, from about 10 to about350 mg, or from about 50 to about 200 mg. The specific amount of thesecond active agent will depend on the specific agent used, the type ofdisease being treated or managed, the severity and stage of disease, andthe amount of the compound of Formula I provided herein and any optionaladditional active agents concurrently administered to the patient. Incertain embodiments, the second active agent is oblimersen (GENASENSE®),GM-CSF, G-CSF, SCF, EPO, taxotere, irinotecan, dacarbazine,transretinoic acid, topotecan, pentoxifylline, ciprofloxacin,dexamethasone, vincristine, doxorubicin, COX-2 inhibitor, IL2, IL8,IL18, IFN, Ara-C, vinorelbine, or a combination thereof.

In certain embodiments, GM-CSF, G-CSF, SCF or EPO is administeredsubcutaneously during about five days in a four or six week cycle in anamount ranging from about 1 to about 750 mg/m²/day, from about 25 toabout 500 mg/m²/day, from about 50 to about 250 mg/m²/day, or from about50 to about 200 mg/m²/day. In certain embodiments, GM-CSF may beadministered in an amount of from about 60 to about 500 mcg/m²intravenously over 2 hours or from about 5 to about 12 mcg/m²/daysubcutaneously. In certain embodiments, G-CSF may be administeredsubcutaneously in an amount of about 1 mcg/kg/day initially and can beadjusted depending on rise of total granulocyte counts. The maintenancedose of G-CSF may be administered in an amount of about 300 (in smallerpatients) or 480 mcg subcutaneously. In certain embodiments, EPO may beadministered subcutaneously in an amount of 10,000 Unit 3 times perweek.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with melphalan anddexamethasone to patients with amyloidosis. In certain embodiments, acompound provided herein, e.g., the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, and steroids can be administered to patients with amyloidosis.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with gemcitabine andcisplatinum to patients with locally advanced or metastatic transitionalcell bladder cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination with asecond active ingredient as follows: temozolomide to pediatric patientswith relapsed or progressive brain tumors or recurrent neuroblastoma;celecoxib, etoposide and cyclophosphamide for relapsed or progressiveCNS cancer; temodar to patients with recurrent or progressivemeningioma, malignant meningioma, hemangiopericytoma, multiple brainmetastases, relapsed brain tumors, or newly diagnosed glioblastomamultiforms; irinotecan to patients with recurrent glioblastoma;carboplatin to pediatric patients with brain stem glioma; procarbazineto pediatric patients with progressive malignant gliomas;cyclophosphamide to patients with poor prognosis malignant brain tumors,newly diagnosed or recurrent glioblastoma multiforms; Gliadel® for highgrade recurrent malignant gliomas; temozolomide and tamoxifen foranaplastic astrocytoma; or topotecan for gliomas, glioblastoma,anaplastic astrocytoma or anaplastic oligodendroglioma.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with methotrexate,cyclophosphamide, taxane, abraxane, lapatinib, herceptin, aromataseinhibitors, selective estrogen modulators, estrogen receptorantagonists, and/or PLX3397 (Plexxikon) to patients with metastaticbreast cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with temozolomide topatients with neuroendocrine tumors.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with gemcitabine topatients with recurrent or metastatic head or neck cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with gemcitabine topatients with pancreatic cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients with coloncancer in combination with ARISA®, avastatin, taxol, and/or taxotere.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with capecitabineand/or PLX4032 (Plexxikon) to patients with refractory colorectal canceror patients who fail first line therapy or have poor performance incolon or rectal adenocarcinoma.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withfluorouracil, leucovorin, and irinotecan to patients with Dukes C & Dcolorectal cancer or to patients who have been previously treated formetastatic colorectal cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withrefractory colorectal cancer in combination with capecitabine, xeloda,and/or CPT-11.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with capecitabine andirinotecan to patients with refractory colorectal cancer or to patientswith unresectable or metastatic colorectal carcinoma.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered alone or in combinationwith interferon alpha or capecitabine to patients with unresectable ormetastatic hepatocellular carcinoma; or with cisplatin and thiotepa topatients with primary or metastatic liver cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withpegylated interferon alpha to patients with Kaposi's sarcoma.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withfludarabine, carboplatin, and/or topotecan to patients with refractoryor relapsed or high-risk acuted myelogenous leukemia.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withliposomal daunorubicin, topotecan and/or cytarabine to patients withunfavorable karotype acute myeloblastic leukemia.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withgemcitabine, abraxane, erlotinib, geftinib, and/or irinotecan topatients with non-small cell lung cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withcarboplatin and irinotecan to patients with non-small cell lung cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered with doxetaxol topatients with non-small cell lung cancer who have been previouslytreated with carbo/VP 16 and radiotherapy.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withcarboplatin and/or taxotere, or in combination with carboplatin,pacilitaxel and/or thoracic radiotherapy to patients with non-small celllung cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withtaxotere to patients with stage IIIB or IV non-small cell lung cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withoblimersen (Genasense®) to patients with small cell lung cancer.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withABT-737 (Abbott Laboratories) and/or obatoclax (GX15-070) to patientswith lymphoma and other blood cancers.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered alone or in combinationwith a second active ingredient such as vinblastine or fludarabine topatients with various types of lymphoma, including, but not limited to,Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma,cutaneous B-Cell lymphoma, diffuse large B-Cell lymphoma or relapsed orrefractory low grade follicular lymphoma.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered in combination withtaxotere, IL-2, IFN, GM-CSF, PLX4032 (Plexxikon) and/or dacarbazine topatients with various types or stages of melanoma.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered alone or in combinationwith vinorelbine to patients with malignant mesothelioma, or stage IIIBnon-small cell lung cancer with pleural implants or malignant pleuraleffusion mesothelioma syndrome.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withvarious types or stages of multiple myeloma in combination withdexamethasone, zoledronic acid, palmitronate, GM-CSF, biaxin,vinblastine, melphalan, busulphan, cyclophosphamide, IFN, palmidronate,prednisone, bisphosphonate, celecoxib, arsenic trioxide, PEG INTRON-A,vincristine, or a combination thereof.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withrelapsed or refractory multiple myeloma in combination with doxorubicin(Doxil®), vincristine and/or dexamethasone (Decadron®).

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withvarious types or stages of ovarian cancer such as peritoneal carcinoma,papillary serous carcinoma, refractory ovarian cancer or recurrentovarian cancer, in combination with taxol, carboplatin, doxorubicin,gemcitabine, cisplatin, xeloda, paclitaxel, dexamethasone, or acombination thereof.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withvarious types or stages of prostate cancer, in combination with xeloda,5 FU/LV, gemcitabine, irinotecan plus gemcitabine, cyclophosphamide,vincristine, dexamethasone, GM-CSF, celecoxib, taxotere, ganciclovir,paclitaxel, adriamycin, docetaxel, estramustine, Emcyt, denderon or acombination thereof.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withvarious types or stages of renal cell cancer, in combination withcapecitabine, IFN, tamoxifen, IL-2, GM-CSF, Celebrex®, or a combinationthereof.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withvarious types or stages of gynecologic, uterus or soft tissue sarcomacancer in combination with IFN, a COX-2 inhibitor such as Celebrex®,and/or sulindac.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withvarious types or stages of solid tumors in combination with celebrex,etoposide, cyclophosphamide, docetaxel, apecitabine, IFN, tamoxifen,IL-2, GM-CSF, or a combination thereof.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withscleroderma or cutaneous vasculitis in combination with celebrex,etoposide, cyclophosphamide, docetaxel, apecitabine, IFN, tamoxifen,IL-2, GM-CSF, or a combination thereof.

Also encompassed herein is a method of increasing the dosage of ananti-cancer drug or agent that can be safely and effectivelyadministered to a patient, which comprises administering to the patient(e.g., a human) or an enantiomer or a mixture of enantiomers thereof, ora pharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof. Patients that can benefit by thismethod are those likely to suffer from an adverse effect associated withanti-cancer drugs for treating a specific cancer of the skin,subcutaneous tissue, lymph nodes, brain, lung, liver, bone, intestine,colon, heart, pancreas, adrenal, kidney, prostate, breast, colorectal,or combinations thereof. The administration of a compound providedherein, e.g., the compound of Formula I, or an enantiomer or a mixtureof enantiomers thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof, alleviates orreduces adverse effects which are of such severity that it wouldotherwise limit the amount of anti-cancer drug.

In one embodiment, a compound provided herein, e.g., the compound ofFormula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered orally and daily in anamount ranging from about 0.1 to about 150 mg, from about 1 to about 50mg, or from about 2 to about 25 mg, prior to, during, or after theoccurrence of the adverse effect associated with the administration ofan anti-cancer drug to a patient. In certain embodiments, a compoundprovided herein, e.g., the compound of Formula I, or an enantiomer or amixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof, isadministered in combination with specific agents such as heparin,aspirin, coumadin, or G-CSF to avoid adverse effects that are associatedwith anti-cancer drugs such as but not limited to neutropenia orthrombocytopenia.

In one embodiment, a compound provided herein, e.g., the compound ofFormula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, is administered to patients withdiseases and disorders associated with or characterized by, undesiredangiogenesis in combination with additional active ingredients,including, but not limited to, anti-cancer drugs, anti-inflammatories,antihistamines, antibiotics, and steroids.

In another embodiment, encompassed herein is a method of treating,preventing and/or managing cancer, which comprises administering thecompound of Formula I, or an enantiomer or a mixture of enantiomersthereof, or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, in conjunction with (e.g.before, during, or after) conventional therapy including, but notlimited to, surgery, immunotherapy, biological therapy, radiationtherapy, or other non-drug based therapy presently used to treat,prevent or manage cancer. The combined use of the compound providedherein and conventional therapy may provide a unique treatment regimenthat is unexpectedly effective in certain patients. Without beinglimited by theory, it is believed that the compound of Formula I mayprovide additive or synergistic effects when given concurrently withconventional therapy.

As discussed elsewhere herein, encompassed herein is a method ofreducing, treating and/or preventing adverse or undesired effectsassociated with conventional therapy including, but not limited to,surgery, chemotherapy, radiation therapy, hormonal therapy, biologicaltherapy and immunotherapy. A compound provided herein, e.g., thecompound of Formula I, or an enantiomer or a mixture of enantiomersthereof, or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, and other active ingredientcan be administered to a patient prior to, during, or after theoccurrence of the adverse effect associated with conventional therapy.

In one embodiment, the compound of Formula I can be administered in anamount ranging from about 0.1 to about 150 mg, from about 1 to about 25mg, or from about 2 to about 10 mg orally and daily alone, or incombination with a second active agent disclosed herein (see, e.g.,section 4.3), prior to, during, or after the use of conventionaltherapy.

In certain embodiments, a compound provided herein, e.g., the compoundof Formula I, or an enantiomer or a mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof, and doxetaxol are administered topatients with non-small cell lung cancer who were previously treatedwith carbo/VP 16 and radiotherapy.

5.6.2 Use with Transplantation Therapy

The compound of Formula I provided herein can be used to reduce the riskof Graft Versus Host Disease (GVHD). Therefore, encompassed herein is amethod of treating, preventing and/or managing cancer, which comprisesadministering the compound of Formula I, or an enantiomer or a mixtureof enantiomers thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof, in conjunctionwith transplantation therapy.

As those of ordinary skill in the art are aware, the treatment of canceris often based on the stages and mechanism of the disease. For example,as inevitable leukemic transformation develops in certain stages ofcancer, transplantation of peripheral blood stem cells, hematopoieticstem cell preparation or bone marrow may be necessary. The combined useof the compound of Formula I provided herein and transplantation therapyprovides a unique and unexpected synergism. In particular, the compoundof Formula I exhibits immunomodulatory activity that may provideadditive or synergistic effects when given concurrently withtransplantation therapy in patients with cancer.

The compound of Formula I can work in combination with transplantationtherapy reducing complications associated with the invasive procedure oftransplantation and risk of GVHD. Encompassed herein is a method oftreating, preventing and/or managing cancer which comprisesadministering to a patient (e.g., a human) the compound of Formula I, oran enantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, before, during, or after the transplantation of umbilical cordblood, placental blood, peripheral blood stem cell, hematopoietic stemcell preparation, or bone marrow. Some examples of stem cells suitablefor use in the methods provided herein are disclosed in U.S. Pat. No.7,498,171, the disclosure of which is incorporated herein by referencein its entirety.

In one embodiment, the compound of Formula I is administered to patientswith multiple myeloma before, during, or after the transplantation ofautologous peripheral blood progenitor cell.

In another embodiment, the compound of Formula I is administered topatients with relapsing multiple myeloma after the stem celltransplantation.

In yet another embodiment, the compound of Formula I and prednisone areadministered as maintenance therapy to patients with multiple myelomafollowing the transplantation of autologous stem cell.

In yet another embodiment, the compound of Formula I and dexamethasoneare administered as salvage therapy for low risk post transplantation topatients with multiple myeloma.

In yet another embodiment, the compound of Formula I and dexamethasoneare administered as maintenance therapy to patients with multiplemyeloma following the transplantation of autologous bone marrow.

In yet another embodiment, the compound of Formula I is administeredfollowing the administration of high dose of melphalan and thetransplantation of autologous stem cell to patients with chemotherapyresponsive multiple myeloma.

In yet another embodiment, the compound of Formula I and PEG INTRO-A areadministered as maintenance therapy to patients with multiple myelomafollowing the transplantation of autologous CD34-selected peripheralstem cell.

In yet another embodiment, the compound of Formula I is administeredwith post transplant consolidation chemotherapy to patients with newlydiagnosed multiple myeloma to evaluate anti-angiogenesis.

In still another embodiment, the compound of Formula I and dexamethasoneare administered as maintenance therapy after DCEP consolidation,following the treatment with high dose of melphalan and thetransplantation of peripheral blood stem cell to 65 years of age orolder patients with multiple myeloma.

In one embodiment, the compound of Formula I is administered to patientswith NHL (e.g., DLBCL) before, during, or after the transplantation ofautologous peripheral blood progenitor cell.

In another embodiment, the compound of Formula I is administered topatients with NHL (e.g., DLBCL) after a stem cell transplantation.

5.6.3 Cycling Therapy

In certain embodiments, the prophylactic or therapeutic agents providedherein are cyclically administered to a patient. Cycling therapyinvolves the administration of an active agent for a period of time,followed by a rest for a period of time, and repeating this sequentialadministration. Cycling therapy can reduce the development of resistanceto one or more of the therapies, avoid, or reduce the side effects ofone of the therapies, and/or improves the efficacy of the treatment.

Consequently, in certain embodiments, the compound of Formula I providedherein is administered daily in a single or divided doses in a four tosix week cycle with a rest period of about a week or two weeks. Thecycling method further allows the frequency, number, and length ofdosing cycles to be increased. Thus, encompassed herein in certainembodiments is the administration of a compound provided herein, e.g.,the compound of Formula I, or an enantiomer or a mixture of enantiomersthereof, or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, for more cycles than aretypical when it is administered alone. In certain embodiments, acompound provided herein, e.g., the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof, is administered for a greater number of cycles that wouldtypically cause dose-limiting toxicity in a patient to whom a secondactive ingredient is not also being administered.

In one embodiment, the compound of Formula I is administered daily andcontinuously for three or four weeks at a dose of from about 0.1 toabout 150 mg/d followed by a break of one or two weeks.

In another embodiment, the compound of Formula I and a second activeingredient are administered orally, with administration of the compoundof Formula I occurring 30 to 60 minutes prior to a second activeingredient, during a cycle of four to six weeks. In certain embodiments,the combination of the compound of Formula I and a second activeingredient is administered by intravenous infusion over about 90 minutesevery cycle. In certain embodiments, one cycle comprises theadministration from about 0.1 to about 150 mg/day of the compound ofFormula I and from about 50 to about 200 mg/m²/day of a second activeingredient daily for three to four weeks and then one or two weeks ofrest. In certain embodiments, the number of cycles during which thecombinatorial treatment is administered to a patient is ranging fromabout one to about 24 cycles, from about two to about 16 cycles, or fromabout four to about three cycles.

5.7 Pharmaceutical Compositions and Dosage Forms

In one embodiment, provided herein are pharmaceutical compositions anddosage forms, which comprise the compound of Formula I, or an enantiomeror a mixture of enantiomers thereof, or a pharmaceutically acceptablesalt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. Inanother embodiment, pharmaceutical compositions and dosage forms furthercomprise one or more excipients.

In certain embodiments, pharmaceutical compositions and dosage formsprovided herein also comprise one or more additional active ingredients.Consequently, pharmaceutical compositions and dosage forms providedherein comprise the compound of Formula I, or an enantiomer or a mixtureof enantiomers thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof, and a secondactive agent. Examples of optional second, or additional, activeingredients are disclosed herein (see, e.g., section 4.3).

Single unit dosage forms provided herein are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), topical (e.g., eye drops or other ophthalmicpreparations), transdermal, or transcutaneous administration to apatient. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; powders;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; eye drops or other ophthalmic preparations suitable fortopical administration; and sterile solids (e.g., crystalline oramorphous solids) that can be reconstituted to provide liquid dosageforms suitable for parenteral administration to a patient.

The composition, shape, and type of dosage forms provided herein mayvary depending on their use. For example, a dosage form used in theacute treatment of a disease may contain larger amounts of one or moreof the active ingredients than a dosage form used in the chronictreatment of the same disease. Similarly, a parenteral dosage form maycontain smaller amounts of one or more of the active ingredients than anoral dosage form used to treat the same disease. See, e.g., Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Whether a particular excipient is suitable for incorporation into apharmaceutical composition or dosage form provided herein depends on avariety of factors, including, but not limited to, the route ofadministration. For example, oral dosage forms such as tablets maycontain excipients not suited for use in parenteral dosage forms. Thesuitability of a particular excipient may also depend on the specificactive ingredients in the dosage form. For example, the decomposition ofsome active ingredients may be accelerated by some excipients such aslactose, or when exposed to water. Active ingredients that compriseprimary or secondary amines are particularly susceptible to suchaccelerated decomposition. Consequently, encompassed herein arepharmaceutical compositions and dosage forms that contain little, ifany, lactose. As used herein, the term “lactose-free” means that theamount of lactose present, if any, is insufficient to substantiallyincrease the degradation rate of an active ingredient.

Lactose-free compositions provided herein can comprise excipients thatare listed, for example, in the U.S. Pharmacopeia (USP) 25-NF20 (2002).In certain embodiments, lactose-free compositions comprise activeingredients, a binder/filler, and a lubricant in pharmaceuticallycompatible and pharmaceutically acceptable amounts. In certainembodiments, lactose-free dosage forms comprise active ingredients,microcrystalline cellulose, pre-gelatinized starch, and magnesiumstearate.

Further encompassed herein are anhydrous pharmaceutical compositions anddosage forms comprising active ingredients, since water can facilitatethe degradation of some compounds. For example, the addition of water(e.g., 5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long-term storage in order to determine characteristics suchas shelf-life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, in certainembodiments, provided herein are anhydrous compositions packaged usingmaterials to prevent exposure to water such that they can be included insuitable formulary kits. Examples of suitable packaging include, but arenot limited to, hermetically sealed foils, plastics, unit dosecontainers (e.g., vials), blister packs, and strip packs.

Encompassed herein are pharmaceutical compositions and dosage forms thatcomprise one or more compounds that reduce the rate by which an activeingredient will decompose. Such compounds, which are referred to hereinas “stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. In certain embodiments, the dosage forms provided hereincomprise the compound of Formula I, or an enantiomer or a mixture ofenantiomers thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof, in an amountranging from about 0.10 to about 1000 mg, from about 0.10 to about 500mg, from about 0.10 to about 200 mg, from about 0.10 to about 150 mg,from about 0.10 to about 100 mg, or from about 0.10 to about 50 mg. Incertain embodiments, the dosage forms provided herein comprise thecompound of Formula I, or an enantiomer or a mixture of enantiomersthereof, or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof, in an amount of about 0.1,about 1, about 2, about 5, about 7.5, about 10, about 12.5, about 15,about 17.5, about 20, about 25, about 50, about 100, about 150, or about200 mg.

5.7.1 Oral Dosage Forms

In certain embodiments, pharmaceutical compositions provided herein thatare suitable for oral administration are formulated as discrete dosageforms, examples of which include, but are not limited to, tablets (e.g.,chewable tablets), caplets, capsules, and liquids (e.g., flavoredsyrups). Such dosage forms contain predetermined amounts of activeingredients and may be prepared by some known methods of pharmacy. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

In certain embodiments, the oral dosage forms provided herein areprepared by combining the active ingredients in an intimate admixturewith at least one excipient according to conventional pharmaceuticalcompounding techniques. Excipients can take a wide variety of formsdepending on the form of preparation desired for administration. Forexample, excipients suitable for use in oral liquid or aerosol dosageforms include, but are not limited to, water, glycols, oils, alcohols,flavoring agents, preservatives, and coloring agents. Examples ofexcipients suitable for use in solid oral dosage forms (e.g., powders,tablets, capsules, and caplets) include, but are not limited to,starches, sugars, microcrystalline cellulose, diluents, granulatingagents, lubricants, binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms may be prepared bysome known methods of pharmacy. In certain embodiments, pharmaceuticalcompositions and dosage forms are prepared by uniformly and intimatelyadmixing the active ingredients with liquid carriers, finely dividedsolid carriers, or both, and then shaping the product into the desiredpresentation if necessary.

In certain embodiments, a tablet is prepared by compression or molding.In certain embodiments, compressed tablets are be prepared bycompressing in a suitable machine the active ingredients in afree-flowing form, e.g., powder or granules, optionally mixed with anexcipient. In certain embodiments, molded tablets are made by molding ina suitable machine a mixture of a powdered compound moistened with aninert liquid diluent.

Examples of excipients that can be used in oral dosage forms providedherein include, but are not limited to, binders, fillers, disintegrants,and lubricants. Binders suitable for use in pharmaceutical compositionsand dosage forms provided herein include, but are not limited to, cornstarch, potato starch, or other starches, gelatin, natural and syntheticgums such as acacia, sodium alginate, alginic acid, other alginates,powdered tragacanth, guar gum, cellulose and its derivatives (e.g.,ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium,sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methylcellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose,(e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixturesthereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105(FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook,Pa.), and mixtures thereof. An specific binder is a mixture ofmicrocrystalline cellulose and sodium carboxymethyl cellulose (e.g.,AVICEL RC-581). Suitable anhydrous or low moisture excipients oradditives include AVICEL-PH-103™ and Starch 1500 LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms provided herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. Incertain embodiments, the binder or filler in pharmaceutical compositionsprovided herein is present in from about 50 to about 99 weight percentof the pharmaceutical composition or dosage form.

Disintegrants are used in the compositions provided herein to providetablets the ability to disintegrate when exposed to an aqueousenvironment. Tablets that contain too much disintegrant may disintegratein storage, while those that contain too little may not disintegrate ata desired rate or under the desired conditions. Thus, a sufficientamount of disintegrant that is neither too much nor too little todetrimentally alter the release of the active ingredients should be usedto form solid oral dosage forms provided herein. The amount ofdisintegrant used varies based upon the type of formulation. In certainembodiments, the pharmaceutical compositions provided herein comprisefrom about 0.5 to about 15 weight percent or from about 1 to about 5weight percent of disintegrant.

Disintegrants that are suitable for use in pharmaceutical compositionsand dosage forms provided herein include, but are not limited to,agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, other starches, clays, other algins, other celluloses, gums, andmixtures thereof.

Lubricants that are suitable for use in pharmaceutical compositions anddosage forms provided herein include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, but are not limited to,a syloid silica gel (AEROSIL200, W.R. Grace Co., Baltimore, Md.), acoagulated aerosol of synthetic silica (Degussa Co. of Plano, Tex.),CAB-O-SIL (a pyrogenic silicon dioxide, Cabot Co. of Boston, Mass.), andmixtures thereof. In certain embodiments, if used at all, lubricants areused in an amount of less than about 1 weight percent of thepharmaceutical compositions or dosage forms into which they areincorporated.

In certain embodiments, provided herein is a solid oral dosage form,comprising the compound of Formula I, or an enantiomer or a mixture ofenantiomers thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof; and one or moreexcipients selected from anhydrous lactose, microcrystalline cellulose,polyvinylpyrrolidone, stearic acid, colloidal anhydrous silica, andgelatin.

In certain embodiments, provided herein is a solid oral dosage form,comprising the compound of Formula I, or an enantiomer or a mixture ofenantiomers thereof, or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, clathrate, or polymorph thereof; and anhydrouslactose, microcrystalline cellulose, polyvinylpyrrolidone, stearic acid,colloidal anhydrous silica, and gelatin.

In certain embodiments, provided herein is a solid oral dosage form,comprising a hydrochloride sale of the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof, or a pharmaceuticallysolvate, hydrate, co-crystal, clathrate, or polymorph thereof; and oneor more excipients selected from anhydrous lactose, microcrystallinecellulose, polyvinylpyrrolidone, stearic acid, colloidal anhydroussilica, and gelatin.

In certain embodiments, provided herein is a solid oral dosage form,comprising a hydrochloride sale of the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof, or a pharmaceuticallysolvate, hydrate, co-crystal, clathrate, or polymorph thereof; andanhydrous lactose, microcrystalline cellulose, polyvinylpyrrolidone,stearic acid, colloidal anhydrous silica, and gelatin.

5.7.2 Delayed Release Dosage Forms

In certain embodiments, the active ingredients provided herein areadministered by controlled release means or by delivery devices.Examples include, but are not limited to, those described in U.S. Pat.Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719, 5,674,533,5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and5,733,566, each of which is incorporated herein by reference in itsentirety. In certain embodiments, such dosage forms are be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Encompassedherein are single unit dosage forms suitable for oral administration,including, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

5.7.3 Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Some suitable vehicles that can be used to provide parenteral dosageforms provided herein include, but are not limited to: Water forInjection USP; aqueous vehicles such as, but not limited to, SodiumChloride Injection, Ringer's Injection, Dextrose Injection, Dextrose andSodium Chloride Injection, and Lactated Ringer's Injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as, but not limited to, corn oil, cottonseed oil, peanut oil,sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms provided herein. For example, cyclodextrin andits derivatives can be used to increase the solubility of a compoundprovided herein, e.g., the compound of Formula I, or an enantiomer or amixture of enantiomers thereof, or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, clathrate, or polymorph thereof. See,e.g., U.S. Pat. No. 5,134,127, the disclosure of which is incorporatedherein by reference in its entirety.

5.7.4 Topical and Mucosal Dosage Forms

Topical and mucosal dosage forms provided herein include, but are notlimited to, sprays, aerosols, solutions, emulsions, suspensions, eyedrops or other ophthalmic preparations, or other forms known to one ofskill in the art. See, e.g., Remington's Pharmaceutical Sciences,16^(th) and 18^(th) eds., Mack Publishing, Easton Pa. (1980 & 1990); andIntroduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,Philadelphia (1985). Dosage forms suitable for treating mucosal tissueswithin the oral cavity can be formulated as mouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide topical and mucosal dosage forms encompassedherein depend on the particular tissue to which a given pharmaceuticalcomposition or dosage form will be applied. With that fact in mind, incertain embodiments, the excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof to form solutions, emulsions or gels, which arenon-toxic and pharmaceutically acceptable. Moisturizers or humectantscan also be added to pharmaceutical compositions and dosage forms ifdesired. Additional examples of such ingredients can be found, e.g., inRemington's Pharmaceutical Sciences, 16^(th) and 18^(th) eds., MackPublishing, Easton Pa. (1980 & 1990).

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

5.7.5 Kits

In certain embodiments, active ingredients provided herein are notadministered to a patient at the same time or by the same route ofadministration. Therefore, encompassed herein are kits which, when usedby the medical practitioner, can simplify the administration ofappropriate amounts of active ingredients to a patient.

In certain embodiments, a kit provided herein comprises a dosage form ofa compound provided herein, e.g., the compound of Formula I, or anenantiomer or a mixture of enantiomers thereof, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof. In certain embodiments, the kit provided herein furthercomprises additional active ingredients, such as oblimersen(GENASENSE®), melphalan, G-CSF, GM-CSF, EPO, topotecan, dacarbazine,irinotecan, taxotere, IFN, COX-2 inhibitor, pentoxifylline,ciprofloxacin, dexamethasone, IL2, IL8, IL18, Ara-C, vinorelbine,isotretinoin, 13 cis-retinoic acid, or a pharmacologically active mutantor derivative thereof, or a combination thereof. Examples of theadditional active ingredients include, but are not limited to, thosedisclosed herein (see, e.g., section 4.3).

In certain embodiments, the kit provided herein further comprises adevice that is used to administer the active ingredients. Examples ofsuch devices include, but are not limited to, syringes, drip bags,patches, and inhalers.

In certain embodiments, the kit provided herein further comprises cellsor blood for transplantation as well as pharmaceutically acceptablevehicles that can be used to administer one or more active ingredients.For example, if an active ingredient is provided in a solid form thatmust be reconstituted for parenteral administration, the kit cancomprise a sealed container of a suitable vehicle in which the activeingredient can be dissolved to form a particulate-free sterile solutionthat is suitable for parenteral administration. Examples ofpharmaceutically acceptable vehicles include, but are not limited to:Water for Injection USP; aqueous vehicles such as, but not limited to,Sodium Chloride Injection, Ringer's Injection, Dextrose Injection,Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as, but not limited to, corn oil, cottonseed oil, peanut oil,sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

6. EXAMPLES

Certain embodiments of the invention are illustrated by the followingnon-limiting examples.

6.1 Preparation of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione

Step 1: To a solution of potassium hydroxide (16.1 g, 286 mmol) in water(500 mL), was added 3-nitrophthalimide (25.0 g, 130 mmol) in portion at0° C. The suspension was stirred at 0° C. for 3 hrs, and then heated to30° C. for 3 hrs. To the solution, was added HCl (100 mL, 6N). Theresulting suspension was cooled to 0° C. for 1 hr. The suspension wasfiltered and washed with cold water (2×10 mL) to give 3-nitro-phthalamicacid as a white solid (24.6 g, 90% yield): ¹H NMR (DMSO-d₆) δ 7.69 (brs,1H, NHH), 7.74 (t, J=8 Hz, 1H, Ar), 7.92 (dd, J=1, 8 Hz, 1H, Ar), 8.13(dd, J=1, 8 Hz, 1H, Ar), 8.15 (brs, 1H, NHH), 13.59 (s, 1H, OH); ¹³C NMR(DMSO-d₆) δ 125.33, 129.15, 130.25, 132.54, 136.72, 147.03, 165.90,167.31.

Step 2: To a mixture of 3-nitro-phthalamic acid (24.6 g, 117 mmol) andpotassium hydroxide (6.56 g, 117 mmol) in water (118 mL), was added amixture of bromine (6 mL), potassium hydroxide (13.2 g, 234 mmol) inwater (240 mL) at 0° C., followed by addition of a solution of potassiumhydroxide (19.8 g, 351 mmol) in water (350 mL). After 5 minutes at 0°C., the mixture was heated in a 100° C. oil bath for 1 hr. The reactionsolution was cooled to room temperature, and then, in an ice-water bathfor 30 minutes. To the mixture, a solution of HCl (240 mL, 2N) was addeddropwise at 0° C., and the resulting mixture was kept for 1 hr. Thesuspension was filtered and washed with water (5 mL) to give2-amino-6-nitro-benzoic acid as yellow solid (15.6 g, 73% yield): HPLC:Waters Symmetry C₁₈, 5 μm, 3.9×150 mm, 1 mL/min, 240 nm, CH₃CN/0.1%H₃PO₄, 5% grad to 95% over 5 min, 5.83 min (85%); ¹H NMR (DMSO-d₆) δ6.90 (dd, J=1, 8 Hz, 1H, Ar), 7.01 (dd, J=1, 9 Hz, 1H, Ar), 7.31 (t, J=8Hz, 1H, Ar), 8.5-9.5 (brs, 3H, OH, NH₂); ¹³C NMR (DMSO-d₆) δ 105.58,110.14, 120.07, 131.74, 149.80, 151.36, 166.30; LCMS: MH=183.

Step 3: A mixture of 2-amino-6-nitro-benzoic acid (1.5 g, 8.2 mmol) inacetic anhydride (15 mL) was heated at 200° C. for 30 minutes in amicrowave oven. The mixture was filtered and washed with ethyl acetate(20 mL). The filtrate was concentrated in vacuo. The solid was stirredin ether (20 mL) for 2 hrs. The suspension was filtered and washed withether (20 mL) to give 2-methyl-5-nitro-benzo[d][1,3]oxazin-4-one as alight brown solid (1.4 g, 85% yield): HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, CH₃CN/0.1% H₃PO₄, 5% grad 95% in 5 min,5.36 min (92%); ¹H NMR (DMSO-d₆) δ 2.42 (s, 3H, CH₃), 7.79 (dd, J=1, 8Hz, 1H, Ar), 7.93 (dd, J=1, 8 Hz, 1H, Ar), 8.06 (t, J=8 Hz, 1H, Ar); ¹³CNMR (DMSO-d₆) δ 20.87, 107.79, 121.54, 128.87, 137.19, 147.12, 148.46,155.18, 161.78; LCMS: MH=207.

Step 4: Two vials each with a suspension of5-nitro-2-methyl-benzo[d][1,3]oxazin-4-one (0.60 g, 2.91 mmol) and3-amino-piperidine-2,6-dione hydrogen chloride (0.48 g, 2.91 mmol) inpyridine (15 mL) were heated at 170° C. for 10 minutes in a microwaveoven. The suspension was filtered and washed with pyridine (5 mL). Thefiltrate was concentrated in vacuo. The resulting mixture was stirred inHCl (30 mL, 1N), ethyl acetate (15 mL) and ether (15 mL) for 2 hrs. Thesuspension was filtered and washed with water (30 mL) and ethyl acetate(30 mL) to give a dark brown solid, which was stirred with methanol (50mL) at room temperature overnight. The suspension was filtered andwashed with methanol to give3-(2-methyl-5-nitro-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione as ablack solid (490 mg, 27% yield). The solid was used in the next stepwithout further purification.

Step 5: A mixture of3-(2-methyl-5-nitro-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione (250mg) and Pd(OH)₂ on carbon (110 mg) in DMF (40 mL) was shaken underhydrogen (50 psi) for 12 hrs. The suspension was filtered through a padof Celite and washed with DMF (10 mL). The filtrate was concentrated invacuo and the resulting oil was purified by flash column chromatography(silica gel, methanol/methylene chloride) to give3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione as awhite solid (156 mg, 69% yield): HPLC: Waters Symmetry C₁₈, 5 μm,3.9×150 mm, 1 mL/min, 240 nm, 10/90 CH₃CN/0.1% H₃PO₄, 3.52 min (99.9%);mp: 293-295° C.; ¹H NMR (DMSO-d₆) δ 2.10-2.17 (m, 1H, CHH), 2.53 (s, 3H,CH₃), 2.59-2.69 (m, 2H, CH₂), 2.76-2.89 (m, 1H, CHH), 5.14 (dd, J=6, 11Hz, 1H, NCH), 6.56 (d, J=8 Hz, 1H, Ar), 6.59 (d, J=8 Hz, 1H, Ar), 7.02(s, 2H, NH₂), 7.36 (t, J=8 Hz, 1H, Ar), 10.98 (s, 1H, NH); ¹³C NMR(DMSO-d₆) δ 20.98, 23.14, 30.52, 55.92, 104.15, 110.48, 111.37, 134.92,148.17, 150.55, 153.62, 162.59, 169.65, 172.57; LCMS: MH=287; Anal.Calcd. for C₁₄H₁₄N₄O₃+0.3H₂O: C, 57.65; H, 5.05; N, 19.21. Found: C,57.50; H, 4.73; N, 19.00.

6.2 Assays 6.2.1 TNFα Inhibition Assay in PMBC

Peripheral blood mononuclear cells (PBMC) from normal donors areobtained by Ficoll Hypaque (Pharmacia, Piscataway, N.J., USA) densitycentrifugation. Cells are cultured in RPMI 1640 (Life Technologies,Grand Island, N.Y., USA) supplemented with 10% AB+human serum (GeminiBio-products, Woodland, Calif., USA), 2 mM L-glutamine, 100 U/mLpenicillin, and 100 μg/mL streptomycin (Life Technologies)).

The PBMCs (2×10⁵ cells) were plated in 96-well flat-bottom Costar tissueculture plates (Corning, N.Y., USA) in triplicate. The cells werestimulated with LPS (from Salmonella abortus equi, Sigma cat. no.L-1887, St. Louis, Mo., USA) at 1 ng/mL final in the absence or presenceof compounds to be tested. The compounds were dissolved in DMSO (Sigma)and further dilutions were done in culture medium immediately beforeuse. The final DMSO concentration in all assays was about 0.25%. Thecompounds were added to cells 1 hour before LPS stimulation. The cellswere then incubated for 18-20 hours at 37° C. in 5% CO₂, andsupernatants were then collected, diluted with culture medium andassayed for TNFα levels by ELISA (Endogen, Boston, Mass., USA). IC₅₀values were calculated using non-linear regression, sigmoidaldose-response, constraining the top to 100% and bottom to 0%, allowingvariable slope (GraphPad Prism v3.02).

6.2.2 Inhibition of MM Cell Proliferation

The ability of compounds to effect the proliferation of MM cell lineswas investigated in an in vitro study. Uptake of [³H]-thymidine by H929MM cells and 7-AAD uptake in several MM cell lines (H929, U266B1,Anbl-6, KMS-34, OPM-2, DF-15, DF15/R, CAG, MM1.S and LP-1) was measuredas an indicator of cell proliferation. Cells were incubated in thepresence of compounds for 72 hours ([³H]-thymidine was included for thelast 6 hours of the incubation period) or 5 days followed by 7-AADuptake to measure and count viable cells.

6.2.3 Cytokine Production by T Cells

T cells were isolated from buffy coat by negative selection using theRosetteSep® T Cell Enrichment Cocktail. The manufacturer's procedureswere followed accordingly. All 96-well plates were pre-coated with 3μg/ml anti-human CD3 antibody in 100 μl 1×PBS for 4 hours at 37° C. Theplates were washed three times with RPMI-1640 Complete Media prior tothe T cell assay. T cells were then plated in CD3 pre-coated plates at adensity of 2.5×10⁵ cells/well in 180 μl RPMI-1640 Complete Media. Thecells were treated with 20 μl 10× titrated compounds at 10, 1, 0.1,0.01, 0.001, 0.0001 and 0.00001 μM. Final DMSO concentrations were0.25%. The plates were incubated for 48 hours at 37° C., 5% CO₂. After48 hours, the supernatants were harvested and tested by a multi-plexcytometric bead array (CBA) assay for the followingcytokines/chemokines: IL-2, IL-3, IL-5, IL-10, IL-13, IL-15, IL-17a,GM-CSF, G-SCF, IFN-γ, TNF-α and RANTES. The CBA plates were analyzed onthe Luminex IS100 instrument.

Cytokine levels were normalized to the amount produced in the presenceof the amount of a compound tested, and EC₅₀ values were calculatedusing non-linear regression, sigmoidal dose-response, constraining thetop to 100% and bottom to 0%, allowing variable slope (GraphPad Prismv3.02).

Anti-CD3-Stimulated Human T Cell Assay

All 96-well plates were pre-coated with 3 μg/mL anti-human CD3 antibodyin 100 μL 1×PBS for 4 hours at 37° C. The plates were washed 3 timeswith RPMI-1640 Complete Media prior to the T cell assay. The T cellswere then plated in anti-CD3-pre-coated plates at a density of 2.5×105cells/well in 180 μL RPMI-1640 Complete Media. The cells were treatedwith 20 μL 10× titrated Celgene compounds at 10, 1, 0.1, 0.01, 0.001,0.0001, and 0.00001 μM in duplicate. The final DMSO concentrations were0.25%. The plates were incubated for 48 hours at 37° C., 5% CO2. After48 hours, the supernatants were harvested and tested by a multiplexcytometric bead array (CBA) assay for the followingcytokines/chemokines: IL-2, IL-3, IL-5, IL-10, IL-13, IL-15, IL-17A,GM-CSF, G-CSF, IFN-γ, TNF-α, and RANTES. The CBA plates were analyzed onthe Luminex IS100 instrument.

6.2.4 Cytotoxicity Assay

Cells line Farage, DOHH2 and Rec-1 were obtained from American TypeCulture Collection (Manassas, Va., USA) Cytotoxicity assays weremeasured in 3 day ATP production assays as follows:

The cells were plated in black/clear-bottom 96-well TC plates (BDFalcon, Cat #353948) at 3000 cells/75 μL (for DoHH-2 and Farage cells)or 6000 cells/75 μL (Rec-1 cells) media per well. Stock solutions (40×)of compounds were prepared in DMSO and 4× solutions were prepared bydiluting the 40× stock solutions 1:10 with 1% DMSO in culture medium Ineach assay plate well, 25 μL of the compound of Formula I in 1% DMSOwere added to the cells in triplicate so that the final volume was 100μL and [DMSO] final was 0.25%. Plates were then sealed with breathablesealing films (ISC BioExpress, Cat # T-2421-50) and placed in a 37° C.,5% CO₂ humidified incubator for 72 hours. In addition, cells were seededin a separate plate in the same manner as above, 25 μL medium in 1% DMSOwas added to each well. This plate was immediately tested in theCellTiter-Glo Luminescent Cell Viability Assay (Promega, Cat # G7572) as0 time point and the results were used to calculate GIC₅₀ in the Farageand DOHH-2 cell experiments

After 72 hours of incubation, 100 μL of CellTiter-Glo reagent were addedto each well and incubated at room temperature with gentle shaking for30 minutes. The plates were then analyzed for luminescence in a TopCountNXT Reader (Packard). Each well was counted for one second. Values forduplicate wells were averaged and then compared to the zero time pointDMSO control (0% inhibition) to calculate the percentage inhibition ofcell growth. Mean DOHH-2 GIC₅₀ values and Farage GIC₅₀ values werecalculated from three experiments. Rec-1 IC₅₀ values were calculatedfrom two experiments.

6.2.5 Cell Cycle Analysis

Cells were treated with DMSO or an amount of a compound provided hereinfor 48 hours. Propidium iodide staining for cell cycle was performedusing CycleTEST PLUS (Becton Dickinson) according to manufacturer'sprotocol. Following staining, cells were analyzed by a FACSCalibur flowcytometer using ModFit LT software (Becton Dickinson).

6.2.6 Apoptosis Analysis

Cells were treated with DMSO or an amount of a compound provided hereinat various time points, then washed with annexin-V wash buffer (BDBiosciences). Cells were incubated with annexin-V binding protein andpropidium iodide (BD Biosciences) for 10 minutes. Samples were analyzedusing flow cytometry.

6.2.7 NK Cell Analysis

Ninety-six-well flat-bottom plates were coated with 100 μg/mL of humanIgG (Sigma) overnight at 4° C. The next day, unbound IgG was washed awaywith cold 1×PBS. NK cells were then plated in the IgG-coated 96-wellplates at 2×105 cells per well in 180 μL RPMI-1640 Media and 10 ng/mL ofrhIL-2 (R & D Systems, MN) was added. Test compounds were added in avolume of 20 μL DMSO. Final concentrations of test compounds were0.0001, 0.001, 0.01, 0.1, 1, or 10 μM. Final DMSO concentrations were0.25%. After 48 hours, the supernatants were harvested and analyzed byELISA for IFN-γ production.

6.2.8 Results

The biological activities of the compound of Formula I are summarized inTables 1 to 5. In the anti-CD3-stimulated human T cell assay describedabove, the compound of Formula I enhanced production of IL-2, IL-3,IL-5, IL-10, IL-15, GM-CSF, INF-γ, RANTES, and TNF-α at concentrationsof 0.01 to 10 μM. Enhancement of IL-2, IL-3, IL-13, GM-CSF, TNF-α, andRANTES by the compound was concentration-dependent. At a concentrationof 0.1 μM of the compound of Formula I, production of IL-2 and IL-13 wasenhanced to levels 14× and 7× those in control cells, respectively. At aconcentration of 1 μM of the compound of Formula I, production of IL-2and IL-13 was enhanced to levels 17× and 8× those in control cells,respectively. The compound enhanced IL-10 production 2-fold at lowconcentrations (≦0.01 μM) but inhibited IL-10 production at 1 and 10 μM.The compound increased IL-5 production 3- and 4-fold at 0.01 and 0.1 μM,respectively, showing less enhancement at both lower and higherconcentrations.

Additionally, it was observed that, in a human umbilical artery assay,the compound of Formula I was a potent anti-angiogenic agent with anIC₅₀ of 9.4 nM; and the compound of Formula I did not inhibit HUVECproliferation.

In a mouse Matrigel™ angiogenesis assay, it was observed that thecompound of Formula I significantly inhibited blood vessel growth at 30mpk and exhibited a dose dependent inhibition of angiogenesis.

It was observed that the compound of Formula I induced G1 arrest inDoHH2 and WSU-DLCL2. It was also observed that, in proliferation assays,the compound of Formula I acted synergistically with Rituxan, ascalculated using the Chou-Talalay method.

In a DoHH2 xenograft model, it was observed that the compound of FormulaI inhibited tumor growth and that the combination of the compound ofFormula I with Rituxan significantly delayed time to tumor endpoint(63%) at 30 mg/kg dose. Tumor growth inhibition was observed at 3 and 30mpk of the compound of Formula I in combination with Rituxan (1 mg/kg),at 45% and 55% on day 12, respectively. It was also observed that thecompound of Formula I significantly inhibited blood vessels counts intumor.

In a WSU-DLCL2 xenograft model, the combination of the compound ofFormula I with Rituxan (2 mg/kg iv qw) yielded 60% and 90% completeregressions (tumor volume<25 mm³) at 3 and 30 mg, respectively.

In a NCI-H929 MM xenograft model, the compound of Formula I inhibitedH929 tumor growth in a dose-dependent manner. On day 19, the compoundshowed 93% tumor growth inhibition at 30 mg/kg, 73% tumor growthinhibition at 3 mg/kg, and 59% tumor growth inhibition at 0.3 mg/kg.

In a U87 GB xenograft model, dose dependent inhibition of tumor volumewas observed. The compound of Formula I significantly inhibited U87tumor growth at 3 and 30 mg/kg qd.

TABLE 1 In Vitro Activities Assay IC₅₀ or EC₅₀ (μM) PBMC TNFα 0.063^(a)WB TNFα 0.164^(a) LPS-induced TNFα 0.017^(a) T cell IL-2 0.012-0.014^(c)REC1 (MCL) 0.47^(a)  DoHH2 (FL) 0.61^(b)  Farage (GCB-DLBCL) 0.70^(b) Human angiogenesis  0.0094^(a) NK cell IFNγ  0.0015^(c) B cellproliferation 0.015^(c) B cell IgG 0.061^(a) Immature MK colonies>10^(a)    Intermediate MK colonies >10^(a)    ^(a)= IC₅₀, ^(b)= GIC₅₀,^(c)= EC₅₀

TABLE 2 In Vitro Activities (5 Day ³H Thymidine Incorporation Assay)IC₅₀ (μM) ABC Subtype OCI-Ly10 0.0085 U2932 0.11-0.12 TMD8 0.44 RIVA 4.3PMBL Karpas-1106P 0.58-0.71 GCB Subtype WSU-DLCL2 0.79-2.1  SUDHL4 >10OCI-Ly19 >10

TABLE 3 Activity of the Compound of Formula I against LenalidomideResistant Cell Lines (IC₅₀ (μM)) H929 D1 1051 1052 1053 1054Lenolidomide 12.64 No IC₅₀ No IC₅₀ No IC₅₀ No IC₅₀ No IC₅₀ (n = 3) Cmpd.of 0.1539 0.3092 2.974 4.238 2.099 6.593 Form. I (n = 3)

TABLE 4 Effect of the Compound of Formula I in HIF- 1α proteinexpression in solid tumor cell Cancer cell lines % Inhibition at (1 μM)Breast Cancer MCF-7 74.82% Colorectal Cancer HCT116 74.60% HT29 78.54%HCT15 69.26% Ovarian Cancer Skov-3 100.00% Ovcar-3 63.39% Prostatecancer DU145 66.01% Pancreatic Cancer Miapaca-3 33.72% Renal Cancer786-O 41.70% Brain Cancer U87 73.81%

TABLE 5 Anti-Proliferative Activity of the Compound of Formula I inDLBCL cell lines Correlation with Anti-proliferative Compound of FormulaI activity (100 nM) Statistics Oncomine ™ ABC scores Correlated P < 0.05r² = 0.48 Oncomine ™ NFκB scores Not Correlated P > 0.05 r² = 0.35Baseline activity of NFκB Correlated P < 0.005 r² = 0.60 subunit p50baseline activity of NFκB Correlated P < 0.01 r² = 0.65 subunit p65Baseline IRF4 gene Correlated P < 0.05 r² = 0.47 expression BaselineSPIB gene Not Correlated P > 0.05 r² = 0.027 expression Baseline cyclinD1 gene Not Correlated P > 0.05 r² = 0.21 expression Baseline A20 geneNot Correlated P > 0.05 r² = 0.044 expression Baseline CARD11 geneCorrelated P < 0.05 r² = 0.54 expression Baseline CRBN gene Correlated P< 0.05 r² = 0.45 expression

6.3 Pharmacokinetics

It was observed that the compound of Formula I had a t_(1/2) of 230 minin human plasma. The oral pharmacokinetic parameters in mouse, rat, andmonkey are summarized in Tables 5 to 7. Exposures (AUC_((0-t))) of thecompound of Formula I increased in a dose proportional manner up to 30mg/kg in SCID mouse, CD-IDS rat, and male monkey. The compound ofFormula I did not inhibit any megakaryocyte progenitor cells at 10 μM.

TABLE 6 Oral Pharmacokinetics SCID Mouse SD Rat Cyno Monkey Dose (mpk) 3po 30 po 3 po C_(max) (ng/mL (μM)) 2900 4800 3300 (10) (17) (11) AUC(ng-h/mL (μM-h) 7100 25000 12000 (25) (87) (43) T_(1/2) (h) 2.7 5.8 CLp(mL/min/kg) 11 1.2 F (%) 53 32

TABLE 7 PK Profiles in Male Monkeys Dose (mg/kg) C_(max) (ng/mL)AUC_(0-t) (ng*h/mL) 0.3 100 (0.36 μM) 1300 (4.5 μM-h) 3 1100 (3.8 μM)14000 (49 μM-h) 10 3100 (11 μM) 38000 (130 μM-h) 30 7700 (27 μM) 99000(350 μM-h)

TABLE 8 Pharmacokinetics in Monkeys on Day 1 Dose (mg/kg) T_(max) (h)C_(max) (ng/mL) AUC₀₋₂₄ (ng*h/mL) 0.15 2 to 4 (m^(a)) 36 (m) 430 (m) 2(f^(b)) 63 (f) 450 (f) 1.5 2 to 4 (m) 510 (m) 4600 (m) 2 (f) 680 (f)4100 (f) 15 4 (m) 4100 (m) 51000 (m) 2 to 4 (f) 4200 (f) 38000 (f)^(a)m: Male; ^(b)f: Female.

TABLE 9 Pharmacokinetics in Monkeys on Day 27 Dose (mg/kg) T_(max) (h)C_(max) (ng/mL) AUC₀₋₂₄ (ng*h/mL) 0.15 4 (m^(a)) 53 (m) 570 (m) 2 to 4(f^(b)) 57 (f) 450 (f) 1.5 2 (m) 560 (m) 5700 (m) 0.5 to 2 (f) 590 (f)4200 (f) 15 2 to 4 (m) 5800 (m) 72000 (m) 4 (f) 7000 (f) 75000 (f)^(a)m: Male; ^(b)f: Female.

The oral administration of the compound of Formula I at 100, 300, and10000 mg/kg/day for 7 consecutive days in the male CD-IGS rat resultedgenerally in near dose proportional exposure increases. The NOAEL wasdetermined to be 1000 mg/kg/day.

6.4 In Vitro DLBCL Cell Thymidine Incorporation Assay

A panel of DLBCL cell lines of various cytogenetic features was testedfor their sensitivity to the antiproliferative activity of the compoundof Formula I (FIG. 2). Cells were treated with the compound of Formula Ifor 5 days at 37° C.; proliferation of cells was determined using³H-thymidine incorporation method. Results of 3 independent experimentsare shown (mean±SD) in FIG. 2. The compound starting at 0.1-1 μMsignificantly (p<0.05) inhibited proliferation of several lines of DLBCLcells, particularly ABC-subtype cells such as Riva, U2932, TMD8, OCI-Ly3and OCI-Ly10 cells. ABC-subtype cells appear more sensitive to theantiproliferative effect than other subtype cells including GCB-DLBCLand PMBL cells.

6.5 Inhibitory Effect on NFκB Activity in DLBCL Cells

DLBCL cells were treated with the compound of Formula I or an IKK1/2dual inhibitor (used as a positive inhibitor control) for 2 days. NF-κBactivity was examined with Active Motif transcription factor assay usingnuclear extracts from cells following treatment. Results are shown inFIG. 1 (mean±SD). The compound of Formula I significantly inhibits NFκBp65 and p50 activity at concentrations of 0.1 μM, 1 μM and 10 μM. Thecompound of Formula I was found to inhibit the NFκB activity in someDLBCL lines of the ABC subtype, such as U2932 and OCI-Ly10 cells. Theseresults suggest that an effect on NFκB signal transduction might beinvolved in the antiproliferative activity of the compound of Formula Iagainst ABC-DLBCL cells, and that the baseline NFκB activity may be apredictive biomarker of lymphoma tumor response to therapy with thecompound.

6.6 In Vivo Mouse Xenograph Model for the OCI-Ly10 Cell Subtype

Efficacy of the compound of Formula I against the OCI-Ly10 cell subtypeis investigated in an in vivo mouse xenograft model. Female CB.17 SCIDmice age 6 to 12 weeks are injected with about 0.2 mL/mouse of 1×10⁷OCI-Ly10 tumor cells in 100% Matrigel sc in flank. Treatment with thecompound of Formula I begins once tumor reaches an average size of 100to 150 mg. Body weight is measured 5/2 and then biweekly to the end ofthe study. Caliper measurement of the tumor is performed biweekly. Theendpoint of the study is tumor growth delay (TGD). The percentage TGD iscalculated. Animals are monitored individually. The endpoint of thestudy is a tumor volume of about 1000 m³ or 60 days, whichever comesfirst. Responders to therapy may be followed longer.

Tumor collection: collect tumors in RNAse free environment (divide into3 parts). Part is is preserved via snap freeze as a powder for futureprotein analysis, shipping condition −80° C. Part 2 is preserved in RNAlater, snap freeze, shipping condition −80° C. Part 3 is preserved informalin for 24 hours, then 70% ethanol, ship at room temperature to PAIfor paraffin embedding. The treatment plan is shown below.

Gr. N Agent mg/kg Route Schedule 1 10 vehicle 1 — po qd x 28 2 10Compound of Formula I 3 po qd x 28 3 10 Compound of Formula I 10 po qd x28 4 10 Compound of Formula I 30 po qd x 28 5 10 vincristine 1 iv q4d x28

6.7 Multiple Myeloma Models

The ability of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione toinhibit cancer cell growth was evaluated in a number of multiple myeloma(MM) cell lines using in vitro and in vivo methods (FIGS. 5A & 5B).3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione wasshown to inhibit MM cell proliferation in a number of cell lines (FIGS.5A, 5B & 6). For example, the antiproliferative effect of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione wasdemonstrated in a N929 xenograft model (FIG. 6).

6.8 Cereblon Models in ABC-DLBCL, Multiple Myeloma and Colorectal CancerCells

The effect of the protein cereblon (CRBN) on the efficacy of thecompound of Formula Ito inhibit the proliferation, cell cycleprogression and/or cell invasion of various cancer cell lines wasstudied. The compound of Formula I was found to interact with endogenousmyeloma CRBN and in a dose-dependent manner. The compound of Formula Ialso interacts with HepG2 HCC CRBN in a dose-dependent manner. Inaddition, the compound of Formula I was found to inhibit CRBNubiquitination with an IC₅₀ of 208.7 μM.

ABC-DLBCL Cell Model

The expression of cereblon was found to modulate the efficacy of thecompound of Formula I against proliferation of ABC-DLBCL cell lines(FIG. 7A-7C). Cereblon was required for inhibition of each of IRF4expression, NFκB activity, and cell proliferation.

Myeloma Cell Models

The effect of cereblon in H929 myeloma cells was also evaluated. H929cells were transfected with mock, negative control siRNA andCRBN-siRNA-7 for 24, 48, 72 and 96 hours. Cells were treated 24 h aftertransfection with DMSO (0.1%) or the compound of Formula I for 1, 2, 3days and the effect on cell cycle and proliferation investigated. Thecompound of Formula I induced a delay of cell cycle progression,measured as the decrease of the number of cells in S phase, in controlmock and negative control siRNA-transfected cells after 72 h treatment(FIG. 8). Knockdown of CRBN markedly abrogated drug-induced delay incell cycle progression in H929 cells from 65 to 22% for the compound ofFormula I.

RT-PCR and Western blot analysis was used to measure the levels of keycell cycle and apoptotic regulators in order to further investigate theeffects of CRBN on the cell cycle arrest induced by the compound ofFormula I. In H929 cells, the cell cycle arrest in G1 phase by thecompound of Formula I coincides with a reduction of tumor suppressor,pRb, phosphorylation and the oncogene and myeloma survival factor IRF4.Western blot analysis showed that the compound of Formula I decreasedphosphorylation of pRB (FIGS. 9A, 9B and 9C) and total level of proteinIRF4 (FIGS. 9D, 9E and 9F). The effect was reduced by knockdown of CRBNsuggesting that inhibition of cell cycle progression by the drugsrequires CRBN protein.

The compound of Formula I was found to inhibit the proliferation ofCRBN-sensitive MM cell lines U266, 100-1 and 1K-2 (FIG. 10).

Colorectal Cell Model

The expression of cereblon also modulates the anti-invasive activity ofthe compound of Formula I in HCT-15 colorectal cancer cells (FIG. 11).The ability of the compound of Formula Ito inhibit invasion of HCT-15cells was reduced by siCRBN.

6.9 Solid Tumor Models

The compound of Formula I was evaluated for its effect on solid tumorcell lines from a variety of histologies (e.g., breast, ovarian,colorectal, HCC). The compound of Formula I inhibits hypoxia-inducedHIF1-α expression in many such solid tumor cell lines (FIG. 12A-12I). Inaddition, the compound of Formula I inhibits the invasion of solid tumorcells to varying degrees (Table 10) and cell colony formation (Table11). The inhibition of solid tumor cell colony formation was studied bya single high concentration treatment of the Compound of formula I (10μM) on day 1, followed by monitoring of cell colony formation over thecourse of 10 to 20 days (Table 11, FIGS. 13A & 13B).

The compound of Formula I inhibits U87 glioiblastoma tumor cell growthat 3 and 30 mg/kg q.d. in a xenograft model (FIG. 14).

TABLE 10 Effects of the Compound of Formula I on Invasion of Solid TumorCells Invasion (IC₅₀) Tumor Cell Type Cell Line (stimulation) Compoundof Formula I hepatocellular HepG2 (VEGF) <0.001 SK-HEP-1 (VEGF) 0.0061glioblastoma SNB-19 (PDGF) 0.16 SF-539 (PDGF) 0.025 U251 (PDGF) 3.7SF-295 (PDGF) 0.24 U87 (PDGF) 0.08 colorectal HCT15 (bFGF) 0.0072

TABLE 11 Effects of the Compound of Formula I in Solid Tumor Cell ColonyFormation % Inhibition of Colony Tumor Cell Type Cell Line Formation^(a)hepatocellular HCT15 3 HCT116  13** Colo-205  17** ovarian OVCAR-3 18*HCC SK-HEP-1 6 HEP-G2   6.9 glioblastoma SF268   0.6 SF295  12.9 U251−6  U87 2 breast MDA-MB-453 −7  MCF-7   1.4 ZR-75-1  90** prostate PC-3 14.8 ^(a)10 μM of Compound of Formula I. *p < 0.5; **p < 0.001 (versusDMSO).

6.10 PBMC Cytokine Profiling

The compound of Formula I was selected for activity profiling of eleven(11) cytokines and chemokines, i.e., interleukin (IL)-1β, IL-6, IL-8,granulocyte macrophage colony stimulating factor (GM-CSF),macrophage-derived chemokine (MDC), macrophage inflammatory protein-1alpha (MIP-1α), macrophage inflammatory protein-1beta (MIP-1β), tumornecrosis factor-alpha (TNF-α), IL-10, monocyte chemotactic protein-1(MCP-1), and RANTES (regulated upon activation normal T-cell expressed,and secreted) using lipopolysaccharide-stimulated human peripheral bloodmononuclear cells (hPBMCs) obtained from 2-6 donors.

The compound of Formula I inhibited the production of (in order ofpotency) TNF-α (IC₅₀=0.034 μM), >IL-1β (IC₅₀=0.054 μM)>IL-6 (IC₅₀=0.060μM)>MDC (IC₅₀=0.062 μM)>MIP-1α (IC₅₀=0.30 μM)>GM-CSF (IC₅₀=0.95 μM)>IL-8(IC₅₀>10 μM)>MIP-1β (IC₅₀>10 μM) (Table 12). The compound of Formula Ialso enhanced IL-10, MCP-1, and RANTES production with mean percent ofcontrol values of 480%, 236%, and 131%, respectively at the 0.1 μMconcentration.

TABLE 12 Summary of Cytokine Inhibitory Profile of the Compound ofFormula I IL-6 IL-8 IL-1β GM-CSF MDC MIP-1α MIP-1 β TNF-α IC₅₀ 0.060 >100.054 0.95 0.062 0.30 >10 0.034

6.11 VEGF-, BFGF-, and HGF-Induced HUVEC Tube Formation, Migration, andInvasion

The compound of formula I demonstrated potent inhibitory activity in anin vitro assay of human umbilical vascular endothelial cell (HUVEC)invasion. The compound of formula I strongly inhibited vascularendothelial growth factor (VEGF)-, basic fibroblast growth factor(bFGF)-, and hepatocyte growth factor (HGF)-induced invasion, weaklyinhibited VEGF and bFGF-induced HUVEC tube formation and migration, andeither enhanced or did not inhibit proliferation of growthfactor-induced HUVEC proliferation. The IC₅₀ value for inhibition ofVEGF-induced HUVEC invasion was 0.29 nM. The IC₅₀ value for inhibitionof bFGF-induced HUVEC invasion was 5.5 nM. The IC₅₀ value for inhibitionof HGF-induced HUVEC invasion was 110 nM. The compound of Formula Iinhibited VEGF- and bFGF-induced migration 38% and 28%, respectively ata concentration of 1 μM.

6.12 Clinical Protocol

A Phase 1a/1b, clinical study to determine the safety, tolerability,pharmacokinetics and efficacy of the the compound of Formula I whenadministered orally to subjects with advanced solid tumors,Non-Hodgkin's lymphoma, or multiple myeloma is provided. Thenon-tolerated dose (NTD), the maximum tolerated dose (MTD) and therecommended phase 2 dose (RP2D) are to be defined in the study. Theeffect of the compound on biomarkers of angiogenesis in pre- and duringtreatment tumor biopsies will be evaluated.

Study Design

The study is designed as a Phase 1a/1b study consisting of two parts:dose escalation (Part A), and dose expansion (Part B). In Part A,subjects will receive single and multiple ascending doses of thecompound of Formula Ito measure pharmacokinetics (PK) and identify themaximum tolerated dose (MTD) and the recommended phase 2 dose (RP2D). Astandard dose (3+3) escalation design (Simon et al., 1997) will be usedto identify initial toxicity. Initial cohorts of three subjects will begiven the compound of Formula I (0.5 mg once daily) in dose incrementsof 100% until the first instance of grade 3 or higher toxicity suspectedto be drug-related in the first cycle, at which point the particularcohort will be expanded to a total of six subjects. This standardescalation schedule will be initiated in order to establish thenon-tolerated dose (NTD) and MTD. Smaller increments and additionalsubjects within a dose cohort may also be evaluated for safety.Approximately 20 to 40 subjects will be treated and evaluated in Part A;however, the total number of subjects in Part A depends on the number ofdose cohorts needed to establish the MTD. A dose will be considered theNTD when 2 or more out of 6 evaluable subjects in a cohort experiencedrug-related dose limiting toxicity (DLT) during Cycle 1. When the NTDis established, dose escalation will stop. The MTD is defined as thelast dose level below the NTD with 0 or 1 out of 6 evaluable subjectsexperiencing DLT during Cycle 1. An intermediate dose (i.e., one betweenthe NTD and the last dose level before the NTD) or additional subjectswithin any dose cohort may be required to more precisely determine theMTD and RP2D.

In Part B, subjects may start dosing at the MTD and/or a lower doselevel based on safety, PK and/or PD data from Part A. Approximately 100subjects (up to 20 per cohort), stratified by tumor type, will betreated and evaluated for safety and antitumor activity after every twocycles of therapy. The dose, doses, or schedule appropriate will also bedetermined. During Part B, safety data will be reviewed regularlyregarding the study continuation, as appropriate.

Study Population

Men and women, 18 years or older, with advanced Solid Tumors (ST),Non-Hodgkin's Lymphoma (NHL), Multiple Myeloma (MM), or advancedunresectable solid tumors, including subjects who have progressed on (ornot been able to tolerate) standard therapy or for whom no standardanticancer therapy exists. Selected tumor types include metastaticbreast cancer (mBC), glioblastoma multiforme (GBM), hepatocellularcarcinoma (HCC), diffuse large B-cell lymphoma (DLBCL), and multiplemyeloma (MM).

Dosing and Length of Study

During the first cycle, only in Part A, each subject will beadministered a single daily dose of the compound of Formula I on Day 1followed by a 48-hour observation and PK sampling period, followed onDay 1 by daily uninterrupted dosing for 28 days (Cycle 1=30 days). Insubsequent Part A cycles, subjects are treated in 28-day cycles withcontinuous dosing from Day 1 to 28. The Compound of Formula I will begiven once or twice a day at a dose of 0.1, 0.5, 1, 2, 4, 5, 7.5, 10,20, 25, or 50 mg in an initial dose. The dose may be of 0.1, 0.5, 1, 2,4, 5, 7.5, 10 mg given once a day. The dose may be 50, 25, or 10 mggiven twice a day. The dose may be adjusted up, or down, from thestarting dose during treatment. As described above, if needed, the drugmay be given in a cyclical manner.

In Part B, subjects receive continuous dosing for 28 days from thebeginning—there is no post initial, single dose 48-hour PK collectionperiod.

Therapy will be discontinued if there is evidence of diseaseprogression, unacceptable toxicity or subject/physician decision tostop. Subjects may continue to receive compound without interruption foras long as they derive benefit as judged by the Investigator.

Enrollment is expected to occur over approximately 24 months. Completionof active treatment and subject follow-up is expected to take anadditional 3-6 months

Study Treatments

Celgene Corporation will supply the compound of Formula I (HCl) as 0.1mg, 0.5 mg, 1 mg and 3 mg capsules for oral administration. The compoundwill be packaged in bottles inside boxes containing drug for 28 days.

In Part A (the dose escalation phase), the dose level will start at 0.5mg once daily after the single PK dose. After the first dose isadministered to the last subject in any cohort, subjects are observedfor at least 30 days before the next higher, protocol-specified dosecohort can begin. Intra subject dose escalation is not permitted unlessapproved by the Safety Review Committee (SRC) which will consist of theprincipal investigator and Celgene's medical monitor.

In Part B, subjects may receive the compound of Formula I at the MTDand/or a lower dose level, based on safety, PK and PD evaluations fromPart A. Approximately 100 subjects (preselected tumor types in groups ofup to 20) will be evaluated for safety and antitumor effects.

Overview of Efficacy Assessments

Subjects will be evaluated for efficacy after every 2 cycles. Theprimary efficacy variable is response. Tumor response will be based onResponse Evaluation Criteria in Solid Tumors (RECIST 1.1), InternationalWorkshop Criteria (IWC) for NHL, International Uniform Response Criteriafor Multiple Myeloma (IURC) (Appendix A, Section or, (18.1 .WorkingGroup for GBM (RANO) Oncology-Responses Assessment for Neuro

Secondary/exploratory endpoints include biomarker measurements in bloodand tumor, histopathologic response and correlations withpharmacogenomic findings. Supplementary efficacy variables (e.g., ECOGperformance status, PET outcomes) will also be examined; in addition,hypovascularization changes will be measured by volume transfer constant(Ktrans) and initial AUC (IAUC) using DCE-MRIs.

Overview of Safety Assessments

The safety variables for this study are adverse events, clinicallaboratory variables, 12-lead ECGs (centrally reviewed), LVEFassessments, physical examinations and vital signs.

Overview of Pharmacokinetic Assessments

The PK profiles of the compound of Formula I and its metabolites will bedetermined from serial blood and urine collections during the firsttreatment cycle. These will be correlated with pharmacodynamic (PD)outcomes where possible.

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the claimed embodiments, and are not intended to limit thescope of what is disclosed herein. Modifications that are obvious topersons of skill in the art are intended to be within the scope of thefollowing claims. All publications, patents, and patent applicationscited in this specification are incorporated herein by reference as ifeach such publication, patent or patent application were specificallyand individually indicated to be incorporated herein by reference.

1-62. (canceled)
 63. A method of treating or managing glioma, comprisingadministering to a patient in need of such treatment or management atherapeutically effective amount of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione,which has the following structure:

or an enantiomer or mixture of enantiomers thereof, or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,clathrate, or polymorph thereof.
 64. The method of claim 63, wherein thecompound is3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. 65.The method of claim 63, wherein the compound is a pharmaceuticallyacceptable salt of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione. 66.The method of claim 63, wherein the glioma is glioblastoma multiforms,glioblastoma, brain stem glioma, poor prognosis malignant brain tumor,malignant glioma, recurrent malignant glioma, anaplastic astrocytoma oranaplastic oligodendroglioma.
 67. The method of claim 63, wherein thecancer is relapsed or refractory.
 68. The method of claim 63, furthercomprising the administration of a therapeutically effective amount ofone or more additional active agents.
 69. The method of claim 68,wherein the additional active agent is selected from the groupconsisting of temozolomide, celecoxib, etoposide, cyclophosphamide,temodar, irinotecan, carboplatin, procarbazine, temozolomide, tamoxifen,and topotecan.
 70. The method of claim 63, wherein3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered in an amount of from about 0.5 to about 50 mg per day. 71.The method of claim 70, wherein the compound is administered in anamount of about 0.5 to about 5 mg per day.
 72. The method of claim 70,wherein the compound is administered in an amount of about 0.5, 1, 2, 4,5, 10, 15, 20, 25 or 50 mg per day.
 73. The method of claim 70, whereinthe compound is orally administered.
 74. The method of claim 70, whereinthe compound is administered in a capsule or tablet.
 75. The method ofclaim 74, wherein the compound is administered in 10 mg or 25 mg of acapsule.
 76. The method of claim 63, wherein the compound isadministered for 21 days followed by seven days rest in a 28 day cycle.77. The method of claim 68, further comprising administering radiationtherapy, hormonal therapy, biological therapy or immunotherapy.